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Phytoremediation and metal speciation in highway soils Padmavathiamma, Prabha Kumari 2010

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PHYTOREMEDIATION AND METAL SPECIATION IN HIGHWAY SOILS by Prabha Kumari Padmavathiamma  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Soil Science)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER)  April 2010  © Prabha Kumari Padmavathiamma, 2010  ABSTRACT Research was conducted to develop a cost effective and environmentally friendly technology to limit the dispersal of metal contaminants from highway traffic in the soil to the surrounding natural environment. The study comprised preliminary field measurements followed by two pot experiments and a field study. The first study evaluated the phytoextraction/ phytostabilisation potential of five plant species: Brassica napus L (rape), Helianthus annuus L. (sunflower), Lolium perenne L (perennial rye grass), Poa pratensis L (Kentucky blue grass) and Festuca rubra L (creeping red fescue) for metals (Cu, Mn, Pb and Zn), in soils with different metal contamination levels. The promising plant species identified were Lolium perenne, Festuca rubra and Poa pratensis. Total soil and plant metal concentrations, as well as the relative metal partitioning in different soil fractions and in plants were determined to provide an estimate of the mobility and potential bioavailability of metals in the soil. The second study evaluated the effectiveness of soil-plant-amendment interaction in immobilising metals in the soil. The amendments included lime, phosphate and compost individually and in combination, and were applied to the plant species: Lolium, Poa and Festuca. Maximum metal immobilisation was achieved in the soil by the combined application of amendments in conjunction with growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. The results obtained from first and second studies were confirmed by conducting field studies. A completely randomized factorial experiment in split plot design with three plant species (Lolium, Poa, and Festuca) individually and in combination, with and without soil amendments was conducted along Highway 17 soil in southwest British Columbia. The influence of root-soil interactions and seasonal influence on the solubility and bioavailability of metals in the soil with and without soil amendments was also evaluated. The best management practices (BMP) developed from the study have the applicability for phytostabilisation of metal contaminated sites and can be suggested as a risk management activity, reducing long-term associated risks.  ii  TABLE OF CONTENTS ABSTRACT .......................................................................................................................ii TABLE OF CONTENTS.................................................................................................iii LIST OF TABLES..........................................................................................................viii LIST OF FIGURES........................................................................................................... x LIST OF ABBREVIATIONS........................................................................................xiii ACKNOWLEDGEMENTS........................................................................................... xiv STATEMENT OF COAUTHORSHIP ......................................................................... xv  1  INTRODUCTION ....................................................................................................... 1 1.1 Statement of the problem ...................................................................................... 1 1.2 Scope and objectives............................................................................................. 4 1.3 Research plan ........................................................................................................ 5 1.3.1. Preliminary investigations.............................................................................. 5 1.3.2. Identifying genera of plants which could be suitable for B.C. climatic conditions ................................................................................. 5 1.3.3. Effect of soil amendments in influencing the plants to immobilize metals in the soil............................................................................................. 6 1.3.4. Field experiments for phytostabilisation at highway site ............................... 7 1.4 Research contributions ......................................................................................... 7 1.5 Dissertation organization...................................................................................... 8 1.6 References .......................................................................................................... 10  2  PHYTOREMEDIATION TECHNOLOGY: HYPER-ACCUMULATION METALS IN PLANTS.............................................................................................. 13 2.1 Introduction......................................................................................................... 13 2.2 Categories of phytoremediation.......................................................................... 18 2.2.1 Phytostabilization ........................................................................................ 19 2.2.2 Phytofiltration............................................................................................... 24 2.2.3 Phytovolatilization........................................................................................ 27 2.2.4 Phytoextraction............................................................................................. 29 2.2.4.1 Types of phytoextraction ....................................................................... 33 2.2.4.2 Successful factors for phytoextraction of heavy metals ........................ 33 2.3. Handling of hazardous plant biomass after phytoremediation ........................... 38 iii  2.4. Conclusions......................................................................................................... 39 2.5 References........................................................................................................... 40 3. PRELIMINARY EXAMINATION OF FIELD MEASUREMENTS OF METAL ACCUMULATION - HEAVY METAL STATUS OF SOILS AND PLANTS PRIOR TO PHYTOREMEDIATION ALONG HIGHWAYS ............ 53 3.1 Introduction......................................................................................................... 53 3.2 Materials and methods ........................................................................................ 54 3.2.1 Site description ............................................................................................. 54 th  3.2.1.1 Trans-Canada Highway & 176 St. Site description ............................. 54 3.2.1.2 Highway 17 site ..................................................................................... 55 3.2.1.3 Background locations ............................................................................ 55 3.2.2 Collection of samples and laboratoratory analysis....................................... 56 3.3 Results and discussion ........................................................................................ 57 3.4 Conclusions......................................................................................................... 61 3.5 References........................................................................................................... 62 4. PHYTOREMEDIATION OF METAL-CONTAMINATED SOIL IN TEMPERATE HUMID REGIONS OF BRITISH COLUMBIA, CANADA ...... 63 4.1 Introduction......................................................................................................... 63 4.2 Materials and methods ........................................................................................ 65 4.2.1 Experimental details ..................................................................................... 65 4.2.2 Bio-metric observations ............................................................................... 67 4.2.3 Laboratory analysis ...................................................................................... 67 4.2.4 Statistical analyses........................................................................................ 67 4.3. Results and discussion ........................................................................................ 68 4.3.1 Metal concentrations in plants...................................................................... 69 4.3.2 Metal content in plants ................................................................................. 71 4.3.3 Metal accumulation characteristics .............................................................. 74 4.3.4 Relationships of metal concentration and bio-metric characters of plants... 76 4.3.5 Relationships of metal content and biomass of plants ................................. 78 4.4 Conclusions and recommendations..................................................................... 80 4.5 References........................................................................................................... 82 4. EXPLORATION OF PHYTOREMEDIATION AND ITS EFFECT ON MOBILITY OF METALS IN SOIL – A FRACTIONATIONSTUDY................ 86 5.1 Introduction......................................................................................................... 86 iv  5.2 Materials and methods ........................................................................................ 88 5.3 Results and discussion ........................................................................................ 90 5.3.1 Physico-chemical properties of soil as influenced by plant growth ............. 91 5.3.2 Metal fractionation in Soils .......................................................................... 93 5.3.3 Metal comparison ......................................................................................... 97 5.3.4 Relationship between soil pH and metal fractions ....................................... 97 5.3.5 Relationship of plant metal concentrations to soil metal fractions .............. 99 5.4 Conclusions and recommendations................................................................... 103 5.5 References......................................................................................................... 104 6  EFFECT OF AMENDMENTS ON PHYTOAVAILABILITY AND FRACTIONATION OF COPPER AND ZINC IN CONTAMINATED SOIL ........................................................................................................................ 108 6.1 Introduction....................................................................................................... 108 6.2 Materials and methods ...................................................................................... 110 6.3 Results and discussion ...................................................................................... 112 6.3.1 Effect of soil amendments on metal concentrations and uptake in plants........................................................................................... 112 6.3.2 Accumulation of Cu and Zn in plants ........................................................ 116 6.3.3 Metal fractionation in the soil .................................................................... 119 6.3.4 Relationship between soil pH and metal fractions ..................................... 122 6.3.5 Relationship of plant metal concentration to soil metal fractions .............. 124 6.3.6 Plant metal concentrations and biometric characteristics........................... 125 6.4 Conclusions and recommendations................................................................... 126 6.5 References......................................................................................................... 128  7. PHYTOAVAILABILITY AND FRACTIONATION OF LEAD AND MANGANESE IN CONTAMINATED SOIL FOLLOWING APPLICATION OF THREE AMENDMENTS ................................................................................ 132 7.1 Introduction....................................................................................................... 132 7.2 Materials and methods ...................................................................................... 133 7.3 Results and discussion ...................................................................................... 135 7.3.1 Metal concentrations and uptake in plants ................................................. 135 7.3.2 Accumulation characteristics of Pb and Mn in plants................................ 137 7.3.3 Pb and Mn fractions in the soils ................................................................. 139 7.3.4 Relationships between soil pH and soil metal fractions............................. 143 v  7.3.5  Relationships between soil metal concentrations and the Enrichment Coefficient .............................................................................. 144 7.3.6 Relationships between soil metal fractions and plant metal concentrations.................................................................................. 145 7.3.7 Relationships between soil properties and metal uptake by plants ............ 146 7.4 Conclusions....................................................................................................... 149 7.5 References......................................................................................................... 151 8. RHIZOSPHERE INFLUENCE AND SEASONAL IMPACT ON PHYTOSTABILISATION OF METALS – A FIELD STUDY ................... 154 8.1 Introduction....................................................................................................... 154 8.2 Materials and methods ...................................................................................... 155 8.2.1 Experiment details...................................................................................... 155 8.2.2 Collection of samples and laboratory analysis ........................................... 156 8.2.3 Statistical analysis ...................................................................................... 157 8.3 Results and discussion ...................................................................................... 157 8.3.1 pH and Electrical Conductivity .................................................................. 158 8.3.2 Metal concentrations in soil ....................................................................... 159 8.3.3 Metal fractionation in soil .......................................................................... 160 8.3.4 Metal concentrations and uptake in plants ................................................. 166 8.3.5 Metal accumulation charecteristics in plants.............................................. 171 8.3.6 Bulk soil vs Rhizosphere soil ..................................................................... 174 8.3.6.1 pH ........................................................................................................ 174 8.3.6.2 Metal Fractionation in RS and BS ....................................................... 175 8.3.6.3 Total metal concentrations in RS and BS ............................................ 178 8.4 Conclusions....................................................................................................... 180 8.5 References......................................................................................................... 181  9. CONCLUSIONS AND RECOMMENDATIONS ................................................ 186 9.1 Conclusions....................................................................................................... 186 9.2. Recommendations and future work .................................................................. 189 9.3. References......................................................................................................... 192  APPENDICES ......................................................................................................... 193 Appendix A - Preliminary studies ............................................................................. 193 Appendix B - Stage I study ....................................................................................... 199 vi  Appendix C - Stage II study ...................................................................................... 201 Appendix D - Stage III study .................................................................................... 206 Appendix E - QA/QC. Abstract of ANOVA – Field Experiment............................. 211 Appendix F - List of publications from thesis .......................................................... 220  vii  LIST OF TABLES Table 2.1  Cost of different remediation technologies .................................................. 15  Table 2.2  Different mechanisms of phytoremediation ................................................. 18  Table 2.3  Approaches to revegetation .......................................................................... 20  Table 2.4  Summary of research results – Phytostabilisation........................................ 22  Table 2.5  Summary of research results – Phytofiltration ............................................. 26  Table 2.6  Effect of typical levels for heavy metals in plants ....................................... 30  Table 2.7(a) Examples of hyperaccumulators and their bioaccumulation potential......... 30 Table 2.7(b) Hyperaccumulators and their bioaccumulation potential ............................. 31 Table 2.8  Recent reports on phytoextraction................................................................ 36  Table 3.1  Basic soil characteristics of Highway soils .................................................. 57  Table 3.2  Soil metal concentrations with distance from the highway (HW1) ............. 58  Table 3.3  Metal concentrations in back ground (BG) sites .......................................... 58  Table 3.4  Root/ Shoot ratio, Enrichment Coefficient and Translocation Factor.......... 61  Table 4.1  Metal concentrations according to British Columbia CSR (Contaminated Sites Regulation) standards and studied soil metal concentrations in the pot study ........................................................... 65  Table 4.2  Key characteristics of the original soil sample............................................. 68  Table 4.3  Metal uptake (µg/pot) by plants (roots, shoots and total) at 90 and 120 DAS ....................................................................................................... 72  Table 4.4  Correlations between metal concentrations (mg/kg) in plants (shoot) at 120 DAS....................................................................................... 73  Table 4.5  Translocation Factor (TF) and Enrichment Coefficient (EC) of metals ...... 75  Table 4.6  Correlations between metal concentrations (mg/kg) in plants and biometric characters at 120 DAS........................................................... 77  Table 5.1  Experimental Program for identification of plant species for Phytostabilisation ......................................................................................... 89  Table 6.1  Experimental Program for soil-plant-amendment interaction.................... 110  Table 6.2  Enrichment Coefficient (EC) and Translocation Factor (TF) in different treatments by plant growth and amendment addition ............................... 117  Table 6.3  % partitioning of Cu and Zn in soils with and without plant growth ......... 120  Table 6.4  Correlation coefficients between soil pH and metal fraction ..................... 124 viii  Table 6.5  Correlations between plant (root and shoot) metal concentrations and soil metal fractions............................................................................... 125  Table 6.6.  Correlation coefficients between plant biometric characters and metal concentrations................................................................................... 126  Table 7.1  Physico chemical properties of the studied soils........................................ 135  Table 7.2  Metal concentrations and metal uptake by the plants (root and shoot) ...... 136  Table 7.3  ECroot, ECshoot and TF for Pb and Mn ......................................................... 138  Table 7.4  % partitioning of Pb and Mn in soils with and without plant growth ........ 140  Table 7.5  Relationship between Enrichment Coefficient (EC) and total soil metal concentration (mg/kg) ...................................................................... 144  Table 8.1  Experimental program for field study ........................................................ 156  Table 8.2  Key soil characteristics before the field experiment .................................. 157  Table 8.3  % metal fractionation in the soil before plant growth ................................ 160  Table 8.4  Metal concentrations in plants (mg/kg)...................................................... 167  Table 8.5  Enrichment Coefficient (ECroot and ECshoot) and Translocation Factor (TF) of metals with and without amendments ................................ 172  ix  LIST OF FIGURES  Figure 2.1  Figure 2.2  Figure 2.3 Figure 2.4 Figure 2.5 Figure 3.1 Figure 3.2 Figure 3.3 Figure 4.1 Figure 4.2  Figure 4.3  Figure 5.1 Figure 5.2  Heavy metal content of road-side soils from (a) Brussels-Ortend, Belgium (Albasel and Cottenie, 1985); (b) Osogobo, Nigeria (Fakayode and Olu-Owolabi, 2003); (c) West bank, Palestine (Swaileh et al., 2004); (d) A31 between Nancy and France (Viard et al., 2004) ............................ 14 Heavy metal content in plants growing on contaminated sites (Yoon et al., 2006). (a) Bahia grass (Paspalum notatum); (b) Wire grass (Gentiana pennelliana); (c) Ticktrefoil (Desmodium paniculatum); (d) Flats edge (Cyperus esculentus); (e) Bermuda grass (Cynodon dactylon)....................................................... 17 Schematic mechanism of phytostabilization ............................................... 19 Schematic mechanism of phytovolatilization ............................................. 27 Schematic mechanism of phytoextraction .................................................. 29 Soil metal concentrations with distance from the highway (HW 17) (mean values±SD, n = 3)............................................................................. 59 Concentration of metals in the plants that spontaneously colonised the study sites (mean values±SD, n = 3) .................................................... 60 Metal concentrations (mg/kg) in moss (Rhytidiadelphus squarrosus) ....... 61 Germination per cent (mean values). Error bars represent means ±S.D for three replicates ................................................................. 69 Metal concentrations in plants at 120 DAS. (a) Cu, (b) Pb, (c) Mn, (d) Zn. (1. LB0, 2. LBA, 3. FB0, 4. FBA, 5. HB0, 6. PB0, 7. PBA, 8. BrB0). Error bars represent means ±S.D. for three replicat.................................... 70 Relationship between metal content in plants and plant biomass (dry weight) at 120 DAS. (a) Root biomass and Cu content in roots. (b) Shoot biomass and Cu content in shoots. (c) Root biomass and Pb content in roots. (d) Shoot biomass and Pb content in shoots................................................ 79 (a) pH and (b) Electrical Conductivity of soils at 90 and 120 DAS. Error bars represent ±S.D of means of three replicates. F-values for pH and Electrical Conductivity are significant at P <0.05..... 92 Metal fractionation (%) in soil by the influence of plant growth at 90 and 120 DAS. n = 3, F-values are significant at P <0.05. LB0 (Lolium B0 soil), LBA (Lolium BA soil), FB0 (Festuca B0 soil), FBA (Festuca BA soil), PB0 (Poa B0 soil), PBA (Poa BA soil), HB0 (Helianthus B0 soil) and BrB0 (Brassica B0 soil)........................................................................ 96  x  Figure 5.3  Figure 5.4 Figure 6.1  Figure 6.2  Figure 6.3  Figure 6.4 Figure 6.5  Figure 6.6  Figure 7.1  Effect of soil pH on soil metal fractions (exchangeable, oxide, organic and residual) at 120 DAS. (a) soil pH and Cu fractions, (b) soil pH and Pb fractions, (c) soil pH and Mn fractions, (d) soil pH and Zn fractions......................... 98 Relationship between plant metal concentrations (in root and shoot) and soil metal fractions ............................................................................. 100 Metal concentration in plants (in root and shoot). (a) Cu concentrations in Lolium. (b) Zn concentrations in Lolium, (c) Cu concentrations in Festuca, (d) Zn concentrations in Festuca, (e) Cu concentrations in Poa, (f) Zn concentrations in Poa. BA - spiked soil, BAL - spiked soil plus lime, BAP - spiked soil plus phosphate, BAO - spiked soil plus compost, BALPO - spiked soil plus lime, phosphate and compost. F significant at P<0.05 for both Cu and Zn................................................................... 113 Soil pH as influenced by plant growth and amendment application. * - F significant at P<0.05. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. ............................................................................. 114 Metal uptake by plants. BA - Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost Mean values, n = 3. F significant at P<0.05.............................................. 116 Relationship between soil metal concentrations and Enrichment Co-efficients (EC) for root and shoot........................................................ 118 Partitioning of Cu and Zn in soil by the effect of amendments and plants BA - Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Mean values, n = 3. F significant at P<0.05.............................................. 121 Relationship between soil pH and metal fractions. (a) soil pH and Cu fractions in Lolium soil, (b) soil pH and Zn fractions in Lolium soil, (c) soil pH and Cu fractions in Festuca soil, (d) soil pH and Zn fractions in Festuca soil, (e) soil pH and Cu fractions in Poa soil, (f) soil pH and Zn fractions in Poa soil..................................................... 123 Mn and Pb fractionation in soils. B0 - Initial soil, BA - Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost ......................... 141 xi  Figure 7.2 Figure 7.3  Figure 7.4  Figure 8.1 Figure 8.2  Figure 8.3  Figure 8.4  Figure 8.5 Figure 8.6  Figure 8.7  Relationship between soil pH and metal fractions in soil (a) soil pH and Pb fractions in the soil. (b) soil pH and Mn fractions in the soil .................................................... 143 Relationship between plant metals and soil metal fractions. (a) Exchangeable Pb and Root Pb, (b) Oxide Pb and Root Pb, (c) Exchangeable Mn and Root Mn, (d) Exchangeable Mn and Shoot Mn, (e) Oxide Mn and Root Mn, (f) Oxide Mn and Shoot Mn...................................................................... 145 Relationship between soil properties and metal uptake by the plants. (a) Pb uptake and soil pH, (b) Mn uptake and soil pH, (c) Pb uptake and % organic matter in the soil, (d) Mn uptake and % organic matter in the soil, (e) Pb uptake and available P in the soil, (f) Mn uptake and available P in the soil .................................................. 147 Seasonal influences on pH and electrical conductivity of soil. T0 – without amendments, T1 – with amendments (lime plus phosphate) ................................................................................ 159 % metal fractionation in soil by the combined influence of plants, amendments and seasons. (a) – Cu, (b) – Pb, (c) – Mn, (d) – Zn. T0 – without amendments, T1 – with amendments. L – Lolium, F- Festuca, P – Poa, C – Combination (Lolium + Festuca + Poa). FS – Fallow soil. Mean values, n = 3. F significant at P<0.05 for all the four metals .......................................... 162 Metal uptake by the plants during three seasons. (a) – Cu uptake, (b) – Pb uptake, (c) – Mn uptake, (d) – Zn uptake. Mean values. n = 3, F significant at P <0.05. L- Lolium, F – Festuca, P – Poa, C- Combination (.Lolium + Festuca + Poa). S – summer, A – autumn, W – winter. T0 – without amendments, T1 – with amendments .................................. 169 ECR (Enrichment Coefficientroot), ECS (Enrichment Coefficientshoot), TF (Translocation Factor) of metals during different seasons. Mean values, n = 3. F significant at P<0.05 .for all the four metals ......................................................................................... 173 pH of Rhizosphere soil and Bulk soil. L – Lolium, F – Festuca, P- Poa, C - Combination. Mean values, n = 3. F significant at P<0.05 ................ 174 Metal fractions in Rhizosphere soil and Bulk soil. (a) Cu, (b) Pb, (c) Mn, (d) Zn. RS – Rhizosphere soil, BS – Bulk soil. T0 – without amendments, T1 – with amendments. Mean values, n = 3. F significant at P<0.05 .............................................. 176 Total metal concentrations in Bulk soil and Rhizosphere soil. RS – Rhizosphere soil, BS – Bulk soil. Mean values, n = 3. F significant at P<0.05 for all the four metals. ............................................................... 178  xii  LIST OF ABBREVIATIONS B0 - Original soil BA - Spiked soil BAL - BA soil + lime BAP - BA soil + phosphate BAO - BA soil + compost BALPO - BA soil + lime + phosphate + compost BMP- Best Management Practices BG - Background BrB0 - Brassica in B0 soil BS - Bulk soil CEC - Cation exchange capacity CSR - Contaminated Sites Regulations DAS - Days after sowing EC - Enrichment Coefficient ECR - Enrichment Coefficient (Root) ECS - Enrichment Coefficient (Shoot) EPA - Environmental Protection Agency FB0 - Festuca in B0 soil FBA - Festuca in BA soil HB0 - Helianthus in B0 soil HW - Highway LB0 - Lolium in B0 soil LBA - Lolium in BA soil MMT - Methyl cyclopentadienyl manganese tricarbonyl PB0 - Poa in B0 soil PBA - Poa in BA soil RD – Relative difference RS - Rhizosphere soil SD - Standard deviation SE – Standard error TEL - Tetraethyl Lead TCH - Trans Canada Highway TF - Translocation Factor xiii  ACKNOWLEDGEMENTS  I express my sincere gratitude and appreciation to Dr. Loretta Li, my supervisor, for accepting me as her student and for her excellent guidance, patience, friendship and support during the course of my studies. I value her wisdom, advice and encouragement. This research project would not have been possible without the support and help of Dr. Les Lavkulich, member of my supervisory committee. I am deeply indebted to him for his guidance, help and support during the course of this research and my entire Ph.D program. Our conversations and his expert advice have shaped and motivated my academic and professional growth. My sincere thanks and heartfelt gratitude to Dr. Art Bomke, member of my supervisory committee, for his valuable input in designing experiments, presentation of data and help throughout my learning journey. I am also deeply grateful to Dr. Peter Jolliffe, member of my supervisory committee for his help, encouragement and critical review of manuscripts. I extend my appreciation to Dr. Tony Kozak for providing necessary help in the statistical analysis of the data. I appreciate the assistance and help I received from Dr. Brent A. Hine, Will and Nicci during the course of this study. I also thank all my colleagues from Soil Science and Civil Engineering for their encouragement and friendship. My special gratitude to my husband Kishore and my son Varun for their unconditional understanding, patience, help and emotional support during the course of this study. I also convey my profound gratitude to NSERC (Natural Sciences and Engineering Research Council of Canada) and the B.C Ministry of Transportation and Infrastructure for providing financial support.  xiv  STATEMENT OF COAUTHORSHIP Results derived from the research of this thesis form the basis of six publications in peerreviewed journals and seven papers in refereed conference proceedings. Formulation of the project proposal, research work involving sample collection, conducting pot experiments, laying out of field experiment, recording phenotypical traits, analysis of soil and plant samples, conducting instrumental analyses using Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer, tabulation of data, statistical analysis of data and preparation of manuscripts were performed by Prabha Kumari Padmavathiamma. Dr. Loretta Li as supervisor helped to develop the research program, reviewed the progress of my work and critically reviewed the manuscripts.  xv  1. INTRODUCTION 1.1 Statement of the problem Metal contamination as a result of human activity is of major concern for ecosystem and human health. A common pathway for metal contamination is through the atmosphere and subsequent deposition onto soil or water. Once the metal contaminants reach the soil or water, physicochemical processes govern the reactions and fate of these toxicants. Transportation systems, notably highways, are a universal concern for their contamination of adjacent right-of-ways. Highway systems transect all forms of landscapes in a diverse array of environments. The fate of transport of related metals along highways has received considerable research attention, but the development of cost-effective, non-destructive remediation techniques needs further consideration. This study focuses on two highway sites in southwestern British Columbia and assesses the levels of contamination arising from highway traffic, the use of commercially available plants for remediation and the dynamics of metal uptake and reactions under a range of seasonal conditions. Soil pollution by metals differs from air or water pollution, because metals persist much longer in soils than in other compartments of the biosphere (Asami, 1984; Alkorta et al., 2004). Also certain metals such as Pb and Cr may not be removable and reside virtually permanently (Kabata-Pendias and Adriano, 1995). Since the residence time of metals in soil is of the order of thousands of years (McGrath, 1987), novel technological approaches are required to remediate excess toxic metals. Apart from vehicular traffic, other sources of metal contaminants in soils include metalliferous mining and smelting sites, metallurgical industries, sewage sludge applications, warfare and military training areas or shooting ranges, waste disposal sites, agricultural fertilizers and electronic industries (Alloway, 1995). Road sediments typically contain elevated levels of metals, which may be mobilized by runoff waters (Sansalone and Buchberger, 1997; Sezgin et al., 2003). Heavy metal contaminants in road sediments are derived from: engine and brake pad wear, (e.g. Cd, Cu, and Ni) (Viklander, 1998; Varrica et al., 2003); lubricants (e.g. Cd, Cu and Zn) (Birch and Scollen, 2003); exhaust emissions, (e.g. Pb and Mn) (Al-Chalabi and Hawker, 2000; Sutherland et al., 2003; Zayed et al., 2003); and tire abrasion (e.g. Zn) (Smolders and  Degryse, 2002). The availability of metals to plants as well as lower food-chain organisms along roadside corridors is a potential concern (Turner et al., 2001). Although the use of leaded gasoline has been suspended in North America and most of the industrialized world, lead continues to be used in approximately 74 countries around the globe, predominantly in subSaharan Africa, the Middle East and most of Asia (Hodes et al., 2003). The use of tetraethyl lead (TEL) as an antiknock compound for gasoline engines until the early 1980s and its subsequent replacement by methyl cyclopentadienyl manganese tricarbonyl (MMT) have led to considerable emissions of Pb and Mn to the environment. Metal contaminants from all these sources accumulate in soils to levels which can cause ecological and human health risks. The behaviour of metals (distribution, chemical forms, bioavailability and micro environmental effects) can be related to various pedogenic features such as soil pH, electrical conductivity, redox potential, organic matter content, cation exchange capacity and other surface properties. These features interact and result in certain processes (e.g. accumulation, removal, translocation and transformations) that operate in the soil over time, leading to differential distribution of metals in various fractions. The distribution of metal fractions govern the mobility/immobility of metals, controlling the off-site migration of the soluble/mobile fraction either to surface water or ground water, where they contaminate drinking water resources and enter the food chain (Adriano et al., 2004). Prolonged and sub-clinical exposure to metals causes various health problems in plants, animals and human beings, including disorders such as cancer, neurological and psychiatric disorders, Parkinson’s disease and kidney failure (Baudouin et al., 2002). Given the high costs associated with conventional (ex-situ) cleanup methods and the large (and growing) number of metal-contaminated sites in Canada, there is considerable interest in developing innovative protocols such as phytoremediation strategies, adapted to the extreme climatic conditions in Canada (Environment Canada, 2003). Phytoremediation involves the use of plants to remove, transfer, stabilize and/or degrade contaminants in soil, sediment and water (Hughes et al. 1997). It is a continuum of various strategies that include both treatment and removal and has the potential to remediate metal contaminated sites, while maintaining the functional and ecological integrity of soil after remediation (Chaney et al.1997, Baker and Brooks 1989; Wang et al, 2006). Phytostabilisation is a feasible and practical remediation strategy for busy contaminated sites. There is no attempt to extract the metals from soil, but to 2  immobilise them. In contrast, phytoextraction removes metals from the soil, but, the disposal of metal-loaded plants is expensive and can lead to risk enhancement. Reducing the environmental impact by holding the metal-pollutants at the source location in non-mobile forms so that they do not interfere with the normal life processes of the vegetated cover is phytostabilisation (Smith and Bradshaw, 1979). Metal mobility and bioavailability can be reduced by growing selected plants or adding various immobilizing agents to the soil, or a combination of both (Smith and Bradshaw, 1979; Simon, 2005, Mench et al., 2000). Therefore the selection of suitable plant species and metal specific soil amendments should be geared towards the maximum partitioning of metals into the immobile fractions in soil. Many published reports are available on the suitabilities of different plant species for phytoextraction and phytostabilisation (Clemente et al., 2006; Conesa et al., 2006; Zhu et al., 2007 etc.) or use of amendments in metal stabilisation (Kumpiene et al., 2007; Simon, 2005; Mench et al., 2000 etc.). Most of the reported studies employed hydroponics, focusing on a single metal and assessing the remediation potential only at one stage of growth (Hamlin and Parker, 2006; Weng et al., 2005; Meyers et al., 2008). Also, there is a lack of research addressing the phytoremediation of roadside soils subjected to multicomponent metal solutions, like those subjected to continuous atmospheric and highway runoff loadings. The land associated with highways in BC constitutes about 12,000 km of roads that are approximately 30 m wide (right-of-way). About 1/3 of the highways are unpaved (comprise millions of kilometres around the world) and support plant and animal life (Precciado and Li, 2006). The challenge is to find economical and efficient methods to effectively limit the dispersal of these contaminants into surface water and ground water and to protect the surrounding natural environments. The present study focuses on phytostabilisation of major metals, i.e. Cu, Pb, Mn, and Zn in highway soils using plants that require minimal harvest and other maintenance. Investigations were based on the chemical protocols for metal accumulation and fractionation in soil (total and selective sequential extraction for metals), entry of metals into the plants, the seasonal impacts on metal dynamics in soil and rhizosphere influence on soil metal chemistry. The study provided extensive information on the mobility/immobility of metals as influenced by plant growth at various stages of growth, accumulation characteristics and translocation properties of metals in different plant species, the use of amendments in complementing the plant effect on metal immobilisation, the influence of pedogenic features on metal mobility/immobility, the root soil interactions on metal dynamics in soil and the seasonal impact on metal speciation in the soil. 3  The study provided guidance towards determining the BMP (Best Management Practices) for stabilisation of metal contaminants (Cu, Pb, Mn and Zn), involving suitable plants and soil amendments and considering seasonal influences and root soil metal interactions in the actual field scenario.  1.2 Scope and objectives The present study was undertaken with the objective of developing an enhanced phytoremediation technology to reduce metal contamination in soils along highways so as to reduce negative environmental impacts. The following tasks were undertaken in support of this objective: a) Identifying genera of plants which could be suitable for BC climatic conditions and soils, and be effective for phytoremediation. Vigorously growing plants in polluted sites were collected and their efficiencies for phytoremediation were compared with known phytoremediating plant species, native to North America (Environment Canada’s database PHYTOREM). b) Growing the identified plants in soils with different metal contamination levels to study the removal of heavy metals at different stages of plant-growth. Factors investigated for each crop included total removal of heavy metals, optimum growth time of the species, biomass production and root response. c) Determining the effect of soil properties such as pH, organic matter content, electrical conductivity and cation exchange capacity in influencing the plants to immobilise metals with and without soil amendments. d) Conducting field experiments on phytostabilisation at a highway site featuring plants and amendments identified as promising from the previous experiments.  4  1.3 Research plan  1.3.1. Preliminary investigations Preliminary studies included characterization of the type and extent of contamination of the study site, identification of native plants and assessment of their metal accumulation characteristics. The concentration of metals (Cu, Pb, Mn and Zn) in the study site, the forms in which they were found, plants that spontaneously colonized the site and their metal partitioning were investigated. Soil samples were collected at horizontal distances of 1, 2, 4, 6, and 8 m from each road side investigated (HW1 and HW17) at two depth intervals, 0-15 cm and 15-30 cm. Plants were collected during winter and summer and their metal accumulation characteristics and translocation properties were studied. Information from this study provided insight for formulating the research program for the subsequent studies. A research paper based on this work is presented in Chapter 3.  1.3.2. Identifying genera of plants which could be suitable for B.C climatic conditions and soils and be effective in phytoremediation This Stage I study involved a systematic and comprehensive effort to assess the phytoremediation potential of five plant species, Lolium perenne L (perennial rye grass), Festuca rubra L (red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape), commonly available in regions with temperate maritime climate, for a highway soil in southwest British Columbia. These species were selected because of their known success in phytoremediation elsewhere and readily available seeds for remediation protocols. Comprehensive pot tests with completely randomized experimental design using five plant species and four metals commonly found in highway roadside soils (i.e. Cu, Pb, Mn and Zn) were carried out under outdoor conditions. The soil used for this research was collected from the back yard of Surrey Fire Hall No. 5, located 1 km north of the intersection of TCH (Trans Canada Highway) with the 176 Street overpass in Surrey, British Columbia. This site was selected since it is located away from major traffic corridor and has minimum anthropogenic disturbance. Soils with three different metal concentrations were studied: (a) B0, the original soil containing 52 mg/kg Cu, 93 mg/kg Pb, 215 mg/kg Mn and 70 mg/kg Zn (b) BA, the original soil spiked with addition of all four metals to give total Cu, Pb, Mn, and Zn concentrations of of 80, 146, 408 and 148 mg/kg, respectively. (c) BC, the original soil spiked to provide total Cu, Pb, 5  Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg, respectively. The research protocol involved: (1) estimating the metal uptake by plants at different growth stages; (2) determining the translocation properties and metal accumulation characteristics of the studied plant species; (3) investigating the distribution of metal fractions in the rhizosphere at two different growth stages under variable multimetal contamination levels; (4) examining the relationship of metal fractions to physico-chemical properties of soil; and (5) assessing the efficiencies of these plants for phytoextraction and phytostabilisation in soils. This study helped to identify the best plant species for phytostabilisation under the climatic and soil conditions of southwest British Columbia. Research papers based on the results are presented in Chapters 4 and 5.  1.3.3. Effect of soil amendments in influencing plants to immobilize metals in soil This Stage II study was conducted to compare the efficiencies of the selected plants (from Stage 1) for phytostabilisation in soil with and without soil amendments. Plant species Lolium perenne L, Festuca rubra L and Poa pratensis L were tested in the presence of three soil amendments (lime, phosphate and compost, both individually and in combination) to assess the effect of soilplant-amendment interaction on phytostabilisation of Cu, Pb, Mn and Zn. The efficiency of treatments to stabilize metals was assessed on the basis of metal speciation in soil, partitioning of metals in plants, and metal uptake by the plants. The effects of soil properties such as pH, CEC, organic matter content and electrical conductivity in influencing the plants to immobilize metals with and without soil amendments were evaluated. The original soil collected from the backyard of Surrey Fire Hall No. 5, near the main highway intersection (HW 1 with 176 street in Surrey, British Columbia) was spiked with addition of all four metals to give total concentrations of Cu, Pb, Mn, and Zn of 80, 146, 408 and 148 mg/kg, respectively. To the spiked soils, amendments such as lime, phosphate and compost were added individually and in combination and plants grown. The study was conducted as a pot experiment in a completely randomized design (CRD) with 18 treatments and three replications. The experiment was performed in the greenhouse during the period, August 2006 to November 2006. Research papers based on these results appear in Chapters 6 and 7.  6  1.3.4. Field experiments for phytostabilisation at a highway site The field study is the Stage III study, and it was undertaken on highway soil (HW 17 NB ramp, Deltaport Way, BC) using the soil amendments of lime and phosphate with three previously identified plant species, Lolium perenne L, Festuca rubra L and Poa pratensis L, both individually and in combination. This research addressed the phytostabilisation of metals along highway soils subjected to multi-metal additions by continuous highway runoff. The research tasks included: (1) quantifying the seasonal extent of metal accumulation in soil and assessing the seasonal impact on the metal speciation in the soil by the influence of soil amendments and different plant species; (2) determining accumulation differences between sampling periods in plant parts and to identify the plant part which accumulates significantly higher amounts of metals seasonally; and (3) assessing the influence of root-soil interactions on metal dynamics. The final outcome of the study helped in the development of a remediation strategy for metals (Cu, Pb, Mn and Zn) involving suitable plants and amendments, incorporating seasonal and rhizosphere influences and maintaining the functional and biological integrity of soil after remediation. A research paper based on this work is presented in Chapter 8.  1.4 Research contributions This project is of significant practical and scientific relevance, since the results allow the effects of plant growth and amendment addition on phytostabilisation of metals in highway soils with different multi-metal contamination levels to be assessed. The results highlight the importance in identifying Best Management Practices (BMP) for phytostabilisation of metals (Cu, Pb, Mn and Zn) along highway soils. Plants suitable for phytoextraction and phytostabilisation of different metal contaminants were identified. The stage of plant growth suitable for maximum metal immobilisation, the seasonal impact on metal partitioning in soil and in plants and the effect of rhizosphere chemistry on metal dynamics were investigated, providing necessary information to formulate guidelines for metal remediation of moderately contaminated acid soils. By phytostabilisation, since the metal contaminants are mostly retained in the below ground portion of the vegetation, the transfer or transport of contaminants to the surrounding environment is lessened, reducing the chances of environmental pollution. Even if the plants die and roots disintegrate, the metal-contaminants still remain inactive in the soil as long as the soil physico chemical characteristics are not altered. Addition of lime as a stabilizing agent at the required 7  dose is environmentally friendly, since it has no harmful effects on either increasing the mobility of metals or the growth of associated ecological partners. Similarly the application of P can convert some of the metals, especially Pb to more immobile forms. The chances of eutrophication by P addition can occur only if P is present in a particular ionic form (mobile) which is unlikely for the soil pH created by lime application. Hence the suggested approach, a containment remediation strategy, not only alleviates existing risks, but also reduces the associated risks of metal effects. Inactivating metal contaminants at the source by using suitable plants and natural soil amendments helps to reduce exposure pathways for metal pollutants and contributes to ecological restoration. It also retains the functional and ecological integrity of soil after remediation. The results from these studies provided sufficient information to formulate specific guidelines for phytostabilisation of metal contaminants (Cu, Pb, Mn and Zn) in a highway soil with multimetal contamination.  1.5 Dissertation organization The dissertation is presented in nine chapters addressing specific aspects of metal remediation in soil-plant systems. Chapter 1 provides a general introduction consisting of the problem statement, scope and objectives, research plan and research contributions of this study. Chapter 2 is the review paper on "Phytoremediation Technology: Hyper-Accumulation Metals in Plants", published in Water, Air, and Soil Pollution. Chapters 3 to 9 are journal papers, each of which is comprised of a brief introduction of the specific study, materials and methods adopted and presentation of the results, discussion and conclusions. Chapter 3 focuses on preliminary examination of field measurements of metal accumulation (Cu, Pb, Mn and Zn) in soils and plants along highway sites and investigated the potential for phytostabilisation of plant species that spontaneously colonised the sites. A version of this 8  chapter was presented at the 9th International Conference on the Biogeochemistry of Trace Elements. (ICOBTE), Beijing, China, 2007 and published in “Biogeochemistry of Trace Elements: Environmental Protection, Remediation and Human Health”. Chapters 4 and 5 focus on the first study, dealing with the identification of plant genera suitable for phytoextraction/phytostabilisation under British Columbia climatic conditions and soils. Chapter 4 describes the absorption characteristics and translocation properties of metals in different plant species at two growth stages, 90 and 120 DAS (days after sowing), published in “International Journal of Phytoremediation”. Chapter 5 deals specifically with the effect of plant growth on soil metal fractionation and total soil metal concentration at two plant growth stages, 90 and 120 DAS, published in “Land Reclamation and Contamination”. Chapters 6 and 7 are focused on the second study, dealing with the effect of addition of soil amendments in modifying the soil properties and influencing the plants to immobilize metals. Chapter 6 describes the effect of plants and soil amendments on Cu and Zn immobilization, accepted for publication in “International Journal of Phytoremediation”. Chapter 7 describes the effect of plants and soil amendments on Pb and Mn immobilization, published in “Bioresource Technology”. Chapter 8 reports the field study. It consists of the seasonal impact on metal accumulation in soil, soil metal fractionation at different seasons, seasonal influence on the metal accumulation and translocation in plants. It also discusses the influence of rhizosphere on soil metal dynamics. Chapter 9 consists of general conclusions and recommendations of the various studies. It is followed by an appendix section (Appendix A to F), which provides additional information on study sites, experimental design, weather data during pot experiments and field study, QA/QC procedures and protocols, abstract of ANOVA for field experiment etc.  9  1.6 References Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142. Alkorta, I., Herna´ndez-Allica, J., Becerril, J. M., Amezaga, I., Albizu, I. and Garbisu, C. (2004). Recent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc, cadmium, lead, and arsenic. Reviews in Environmental Science and Bio Technology, 3, 71–90. Alloway, B. J. (1995). Soil processes and the behavior of metals. In: Alloway B. J. (Ed), Heavy metals in soils. London: Blackie, pp. 38–57. Al-Chalabi, A. S. and Hawker, D. (2000). Distribution of vehicular lead in roadside soils of major roads of Brisbane, Australia. Water, Air, and Soil Pollution, 118, 299–310. Asami, T. (1984). Pollution of soils by cadmium in changing metal cycles and human health. Ed. J O Nriagu Dahlem Konferezen, Berlin, Heidelberg, New York, Tokyo, Springer-Verlag. Baker, A. J. M. and Brooks, R. R. (1989). Terrestrial higher plants which hyper accumulate metallic elements – Review of their distribution, ecology, and phytochemistry. Biorecovery, 1, 81–126. Baudouin, C., Charveron, M., Tarrouse, R. and Gall, Y. (2002). Environmental pollutants and skin cancer. Cell Biology and Toxicology, 18, 341–348. Birch, G. E. and Scollen, A. (2003). Heavy metals in road dust, gully pots and parkland soils in a highly urbanised subcatchment of Port Jackson, Australia. Australian Journal of Soil Research, 41, 1329–1342. Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P. and Angle, J. S. (1997). Phytoremediation of soil metals. Current Opinion in Biotechnology, 8, 279–283. Clemente, R., Almela, C. and Bernal, P. M. (2006). A remediation strategy based on active phytoremediation followed by natural attenuation in a soil contaminated by pyrite waste. Environmental Pollution, 143(3), 397-406. Conesa, H. M., Faz, Á. and Arnaldos, R. (2006). Heavy metal accumulation and tolerance in plants from mine tailings of the semiarid Cartagena-La Unión mining district (SE Spain). Science of The Total Environment, 366, 1–11. Environment Canada. (2003). Phytorem – Potential Green solutions for metal contaminated sites, green biotechnology, CD - rom. Hamlin, R. L. and Parker, A. V. (2006). Phytoextraction potential of Indian mustard at various levels of zinc exposure. Journal of Plant Nutrition, 29, 1257–1272.  10  Hodes, G., Thomas, V. and Williams, A. (2003). A Strategy to Phase-Out Lead in African Gasoline. Renewable Energy for Development, Stockholm Environment Institute, 16(3). Hughes, J. B., Shanks, J., Vanderford, M., Lauritzen, J. and Bhadra, R. (1997) Transformation of TNT by aquatic plants and plant tissue cultures. Environmental Science and Technology, 31, 266–271. Kabata-Pendias, A. and Adriano, D. C. (1995). Trace metals in soil amendments and environmental quality, eds. J. E Rechcigl Lewis Publishers, New York, pp. 139-167. Kumpiene, J., Lagerkvist, A. and Maurice, C. (2007). Stabilization of Pb- and Cu-contaminated soil using coal fly ash and peat. Environmental Pollution, 145, 365-373. McGrath, S. P. (1987). Long-term studies of metal transfers following applications of sewage sludge. In Pollutant Transport and Fate in Ecosystems. Eds. P. J Coughtrey, M. H Martin and M. H Unsworth. Special Publication No. 6 of the British Ecological Society, Blackwell Scientific, Oxford, pp. 301–317. Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W. and Edwards, R. (2000): In situ metal immobilization and phytostabilization of contaminated soils. In: Terry N., Bañuelos G. (eds.): Phytoremediation of Contaminated Soil and Water. Lewis Publ., Boca Raton, London, New York, Washington D.C, pp. 323–358. Meyers, D. E. R., Auchterlonie, G. J., Webb, R. I. and Wood, B. (2008). Uptake and localisation of lead in the root system of Brassica juncea. Environ. Pollut., 153 (2), 323-332. Sansalone, J. J. and Buchberger, S. G. (1997). Partitioning and first flush of metals in urban roadway storm water. J. Environ. Eng., 123, 134–143. Sezgin, N., Ozcan, H. K., Demir, G., Nemlioglu, S. and Bayat, C. (2003). Determination of heavy metal concentrations in street dusts in Istanbul E-5 highway. Environment International, 29, 979–985. Simon, L. (2005) Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300. Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612. Smolders, E. and Degryse, F. (2002). Fate and effect of zinc from tire debris in soil. Environmental Science and Technology, 36, 3706- 3710. Sutherland, R. A., Day, J. P. and Bussen, J. O. (2003). Lead concentrations, isotope ratios and source apportionment in road deposited sediments, Honolulu, Oahu, Hawaii. Water, Air, and Soil Pollution, 142, 165-186. Turner, R. E., Swenson, E. M. and. Milan, C. S. (2001). Organic and inorganic contributions to vertical accretion in salt marsh sediments, In M. Weinstein and K. Kreeger [eds.], Concepts and controversies in tidal marsh ecology. Kluwer, pp. 583–595. 11  Varrica, D., Dongarra, G., Sabatino, G. and Monna, F. (2003). Inorganic geochemistry of roadway dust from the metropolitan area of Palermo, Italy. Environmental Geology, 44, 222-230. Viklander, M. (1998). Particle size distribution and metal content in street sediments. Journal of Environmental Engineering, 124, 761-766. Wang, A. S., Angle, J. S., Chaney, R. L., Delorme, T. A. and Reeves, R. D. (2006). Soil pH effects on uptake of Cd and Zn by Thlaspi caerulescens. Plant and Soil, 281(1–2), 325– 337. Weng, G., Wu, L., Wang, Z., Luo, Y. and Christie, P. (2005). Copper uptake by four Elsholtzia ecotypes supplied with varying levels of copper in solution culture. Environment International, 31 (6), 880-884. Zayed, J., Guessous, A., Lambert, J., Carrier, G. and Philippe, S. (2003). Estimati.on of annual Mn emissions from MMT source in the Canadian environment and the Mn pollution index in each province. Science of The Total Environment, 312(1-3), 147-154. Zhu, Y., Yu, H., Wang, J., Fang, W., Yuan, J. and Yang, Z. (2007). Heavy metal accumulations of 24 asparagus bean cultivars grown in soil contaminated with Cd alone and with multiple metals (Cd, Pb, and Zn). J. Agric. Food Chem., 55, 1045-1052. `  12  2.  1  PHYTOREMEDIATION TECHNOLOGY: HYPER-ACCUMULATION METALS  IN PLANTS  2.1 Introduction Heavy metals are ubiquitous environmental contaminants in industrialized societies. Soil pollution by metals differs from air or water pollution, because metals persist in soil much longer than in other compartments of the biosphere (Lasat, 2002). Over recent decades, the annual worldwide release of metals reached 22,000 t (metric ton) for cadmium, 939,000 t for copper, 783,000 t for lead and 1,350,000 t for zinc (Singh et al., 2003). Sources of metal contaminants in soils include metalliferous mining and smelting, metallurgical industries, sewage sludge treatment, warfare and military training, waste disposal sites, agricultural fertilizers and electronic industries (Alloway, 1995). For example, mine tailings rich in sulphide minerals may form acid mine drainage (AMD) through reaction with atmospheric oxygen and water, and AMD contains elevated levels of metals that could be harmful to animals and plants (Stoltz, 2004). Ground-transportation also causes metal contamination. Highway traffic, maintenance, and de-icing operations generate continuous surface and groundwater contaminant sources. Tread ware, brake abrasion, and corrosion are well documented heavy metal sources associated with highway traffic (Ho and Tai, 1988; Fatoki, 1996; Garcia and Millan, 1998; SanchezMartin et al., 2000). Heavy metal contaminants in roadside soils originate from engine and brake pad wear (e.g. Cd, Cu, and Ni) (Viklander, 1998); lubricants (e.g. Cd, Cu and Zn) (Birch and Scollen, 2003, Turer et al., 2001); exhaust emissions, (e.g. Pb) (Gulson et al., 1981; Al-Chalabi and Hawker, 2000; Sutherland et al., 2003); and tire abrasion (e.g. Zn) (Smolders and Degryse, 2002). The concentration ranges of metals of greatest importance in roadside soils are given in Figure 2.1.  1  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2007) Phytoremediation Technology: Hyper-accumulation metals in plants. Water Air Soil Pollution, 184: 105-126. 13  100 80 60 40 20 0 0  Concentration (mg/kg) ..  Concentration (mg/kg) .  140 120  100  Mn Zn Pb Pow er (Mn) Pow er (Pb) Pow er (Zn)  (a)  5 10 Distance from the Highway (m)  350  Pb Cu Zn Pow er (Pb) Pow er (Cu) Pow er (Zn)  (c)  300 250  (b) 75  200 150 100 50 0  Pb Cu Zn Pow er (Pb) Pow er (Zn) Pow er (Cu)  50 25 0 0  15  Concentration (mg/kg) ..  Concentration (mg/kg) .  160  10 20 30 40 50 Distance from the Highway (m)  350  (d)  300 250  60  Pb Zn Pow er (Pb) Pow er (Zn)  200 150 100 50 0  0  5 10 15 20 25 30 35 Distance from the Highway (m)  Figure  Metal  0  50 100 150 200 250 300 350 Distance from the highway (m)  Regression Equation, y 0.1919  Correlation, R  (a)  Mn Zn Pb  64.487x 25.616x-0.427 31.996x-0.0989  0. 495 0.449 0.189  (b)  Pb Cu Zn  347.5x-0.8549 43.347x-0.3368 110.66x-0.3295  0.983 0.981 0.998  (c)  Pb Cu Zn  319.69x-1.1831 197.25x-1.0689 271.6x-0.6321  0.909 0.925 0.897  (d)  Pb Zn  206.93x-0.6 227.69x-0.1842  0.986 0.871  Figure 2.1 Heavy metal content of road-side soils from (a) Brussels-Ortend, Belgium (Albasel and Cottenie, 1985); (b) Osogobo, Nigeria (Fakayode and Olu-Owolabi, 2003); (c) West bank, Palestine (Swaileh et al., 2004); (d) A31 between Nancy and France (Viard et al., 2004). 14  Toxic heavy metals cause DNA damage, and their carcinogenic effects in animals and humans are probably caused by their mutagenic ability (Knasmuller et al., 1998; Baudouin et al., 2002). Exposure to high levels of these metals has been linked to adverse effects on human health and wildlife. Lead poisoning in children causes neurological damage leading to reduced intelligence, loss of short term memory, learning disabilities and coordination problems. The effects of arsenic include cardiovascular problems, skin cancer and other skin effects, peripheral neuropathy (WHO, 1997) and kidney damage. Cadmium accumulates in the kidneys and is implicated in a range of kidney diseases (WHO, 1997). The principal health risks associated with mercury are damage to the nervous system, with such symptoms as uncontrollable shaking, muscle wasting, partial blindness, and deformities in children exposed in the womb (WHO, 1997). Metal-contaminated soil can be remediated by chemical, physical or biological techniques (McEldowney et al., 1993). Chemical and physical treatments irreversibly affect soil properties, destroy biodiversity and may render the soil useless as a medium for plant growth. These remediation methods can be costly. Table 2.1 summarizes the cost of different remediation technologies. Table 2.1. Cost of different remediation technologies (Glass, 1999).  Process  Cost($/ton)  Other factors  Vitrification  75-425  Long-term monitoring  Land filling  100-500  Transport/excavation/monitoring  Chemical treatment  100-500  Recycling of contaminants  Electrokinetics  20-200  Monitoring  Phytoextraction  5-40  Disposal of phytomass  Among the listed remediation technologies, phytoremediation is one of the lowest cost techniques for contaminated soil remediation. There is a need to develop suitable cost-effective biological soil remediation techniques to remove contaminants without affecting soil fertility. Phytoremediation could provide sustainable techniques for metal remediation. This paper  15  summarizes the development of phytoremediation for metals in the past two decades. Phytoremediation involves the use of plants to remove, transfer, stabilize and/or degrade contaminants in soil, sediment and water (Hughes et al., 1997). The idea that plants can be used for environmental remediation is very old and cannot be traced to any particular source. The concentration of metal uptake in plants is shown in Figure 2.2. A series of fascinating scientific discoveries, combined with interdisciplinary research, has allowed phytoremediation to develop into a promising, cost-effective, and environmentally friendly technology. The term phytoremediation ("phyton" meaning plant, and the Latin suffix "remedium" meaning to clean or restore) refers to a diverse collection of plant-based technologies that use either naturally occurring, or genetically engineered, plants to clean contaminated environments (Cunningham et al., 1997; Flathman and Lanza, 1998). Some plants which grow on metalliferous soils have developed the ability to accumulate massive amounts of indigenous metals in their tissues without symptoms of toxicity (Reeves and Brooks, 1983; Baker and Brooks, 1989; Baker et al., 1991; Entry et al., 1999). The idea of using plants to extract metals from contaminated soil was re-introduced and developed by Utsunamyia (1980) and Chaney (1983). The first field trial on Zn and Cd phytoextraction was conducted by Baker et al. (1991). Several comprehensive reviews have been written, summarizing many important aspects of this novel plant-based technology (Salt et al., 1995, 1998; Chaney et al., 1997; Raskin et al., 1997; Chaudhry et al., 1998; Wenzel et al., 1999; Meagher, 2000; Navari-Izzo and Quartacci, 2001; Lasat, 2002; McGrath et al., 2002; McGrath and Zhao, 2003; McIntyre, 2003; Singh et al., 2003; Garbisu and Alkortha, 2001; Prasad and Freitas, 2003; Alkortha et al., 2004; Ghosh and Singh, 2005; Pilon-Smits, 2005). These reviews give general guidance and recommendations for applying phytoremediation, highlighting the processes associated with applications and underlying biological mechanisms. The present review is intended to give an updated, more concise version of information so far available with respect to different subsets of phyoremediation. It provides a critical overview of the present state of the art, with particular emphasis on phytoextraction and phytostabilization of soil heavy metal contaminants.  16  1600  1200  (a)  1200 1000 800 600 400 0  600 400  0  Pb  Cu Heavy metals  Zn  Pb  Cu Heavy metals  Zn  350  250 (c)  150 100 50  (d)  300 Concentration (mg/kg)  200 Concentration (mg/kg)  800  200  200  250 200 150 100 50 0  0 Pb  Concentration (mg/kg)  (b)  1000 Concentration (mg/kg)  Concentration (mg/kg)  1400  Cu Heavy metals  2000 1800 1600 1400 1200 1000 800 600 400 200 0  Zn  Pb  Cu Heavy metals  Zn  (e) Shoot Root Soil  Pb  Cu  Zn  Heavy metals  Figure 2.2 Heavy metal content in plants growing on contaminated sites (Yoon et al., 2006). (a) Bahia grass (Paspalum notatum); (b) Wire grass (Gentiana pennelliana); (c) Ticktrefoil (Desmodium paniculatum); (d) Flats edge (Cyperus esculentus); (e) Bermuda grass (Cynodon dactylon). .  17  2.2. Categories of phytoremediation Depending on the contaminants, the site conditions, the level of clean-up required, and the types of plants, phytoremediation technology can be used for containment (phytoimmobilization and phytostabilization) or removal (phytoextraction and phytovolatilization) purposes (Thangavel and Subhuram, 2004). The four different plant-based technologies of phytoremediation, each having a different mechanism of action for remediating metal-polluted soil, sediment, or water: (1) phytostabilization, where plants stabilize, rather than remove contaminants by plant root metal retention; (2) phytofiltration, involving plants to clean various aquatic environments; (3) phytovolatilization, utilizing plants to extract certain metals from soil and then release them into the atmosphere by volatilization; and (4) phytoextraction, in which plants absorb metals from soil and translocate them to harvestable shoots where they accumulate. The different mechanisms of phytoremediation are summarized in Table 2.2. Ecological issues also need to be evaluated when developing a phytoremediation strategy for a polluted site. In particular, one has to consider how the phytoremediation efforts might affect local ecological relationships, especially those involving other crops. Since the phytoremediation plants will be grown under contaminated soil/ water conditions, where other crops may not thrive because of contaminant toxicities, the competition problem is unlikely to arise. Table 2.2 Different mechanisms of phytoremediation (Ghosh and Singh, 2005)  Process  Mechanisms  Contaminant  Phytofiltration  Rhizosphere accumulation  Organics/ Inorganic  Phytostabilisation  Complexation  Inorganic  Phytoextraction  Hyper accumulation  Inorganic  Phytovolatilization  Volatilisation by leaves  Organics/ Inorganic  18  2.2.1 Phytostabilization Phytostabilization uses certain plant species to immobilize contaminants in soil, through absorption and accumulation by roots, adsorption onto roots or precipitation within the root zone and physical stabilization of soils. The schematic mechanism of phytostabilization is illustrated in Figure 2.3. This process reduces the mobility of contaminants and prevents migration to groundwater or air. This can re-establish a vegetative cover at sites where natural vegetation is lacking due to high metal concentrations (Tordoff et al., 2000). Thorough planning is essential for successful revegetation, including physical and chemical analyses, bioassays and field trials. The main approaches to revegetation are summarized in Table 2.3. Metal-tolerant species may be used to restore vegetation to such sites, thereby decreasing the potential migration of contaminants through wind, transport of exposed surface soils, leaching of soil and contamination of groundwater (Stoltz and Greger, 2002).  Figure 2.3  Schematic mechanism of phytostabilization.  Unlike other phytoremediative techniques, phytostabilization is not intended to remove metal contaminants from a site, but rather to stabilize them by accumulation in roots or precipitation within root zones, reducing the risk to human health and the environment. It is applied in situations where there are potential human health impacts, and exposure to substances of concern can be reduced to acceptable levels by containment. The disruption to site activities may be less than with more intrusive soil remediation technologies. 19  Phytostabilization is most effective for fine-textured soils with high organic matter content, but it is suitable for treating a wide range of sites where large areas are subject to surface contamination (Cunningham et al., 1997; Berti and Cunningham, 2000). However, some highly contaminated sites are not suitable for phytostabilization, because plant growth and survival is impossible (Berti and Cunningham, 2000). Phytostabilization has advantages over other soil-remediation practices in that it is less expensive, easier to implement, and preferable aesthetically. (Berti and Cunningham, 2000; Schnoor, 2000). When decontamination strategies are impractical because of the extent of the contaminated area or the lack of adequate funding, phytostabilization is advantageous (Berti and Cunningham, 2000). It may also serve as an interim strategy to reduce risk at sites where complications delay the selection of the most appropriate technique. Table 2.3. Approaches to revegetation (adapted from Williamson and Johnson, 1981)  Soil characteristics  Reclamation technique  Problems encountered  Low toxicity- total metal content <0.1%.  Amelioration and direct seeding with grasses and legumes. Seed or transplant ecologically adapted native species. Apply lime, organic matter and fertilizers as necessary  High toxicity-total metal content > 0.1%.  Amelioration and direct seeding with metal tolerant and salt tolerant (saline) ecotypes. Apply lime, organic matter and fertilizers as necessary. Amelioration with 10-50 cm of innocuous mineral waste and organic material and seeding with grasses and legumes. Apply lime and fertilizer if necessary Isolation; surface treatment with 30-100 cm of innocuous barrier material and surface banding with10-30 cm of rooting medium. Apply lime and fertilizer if necessary.  Medium or long –term maintenance program. Expertise required on the characteristics of native flora. Grazing must be strictly monitored and excluded in some situations Commitment to regular management. Expertise required for the selection of tolerant ecotypes. Grazing management not possible. Regression will occur if depths of amendment are shallow or if upward movement of metals occurs. Availability and transport costs limiting. High cost and potential limitation of material availability.  Extreme toxicity  20  Characteristics of plants appropriate for phytostabilization at a particular site include: tolerance to high levels of the contaminant(s) of concern; high production of root biomass able to immobilize these contaminants through uptake, precipitation, or reduction; and retention of applicable contaminants in roots, as opposed to transfer to shoots, to avoid special handling and disposal of shoots. Yoon et al. (2006) evaluated the potential of 36 plants (17 species) growing on a contaminated site and found that plants with a high bio-concentration factor (BCF, metal concentration ratio of plant roots to soil) and low translocation factor (TF, metal concentration ratio of plant shoots to roots) have the potential for phytostabilization (Figure 2.2, a-e). The lack of appreciable metals in shoot tissue also eliminates the necessity to treat harvested shoot residue as a hazardous waste (Flathman and Lanza, 1998). In a field study, mine wastes containing copper, lead, and zinc were stabilized by grasses (Agrostis tenuis cv. Goginan for acid lead and zinc mine wastes, Agrostis tenuis cv. Parys for copper mine wastes, and Festuca rubra cv. Merlin for calcareous lead and zinc mine wastes) (Smith and Bradshaw, 1992). The research of Smith and Bradshaw (1992) led to the development of two cultivars of Agrostis tenuis Sibth and one of Festuca rubra L which are now commercially available for phytostabilizing Pb-, Zn-, and Cu-contaminated soils. Stabilization also involves soil amendments to promote the formation of insoluble metal complexes that reduce biological availability and plant uptake, thus preventing metals from entering the food chain (Adriano et al., 2004; Berti and Cunningham, 2000; Cunningham et al., 1997). One way to facilitate such immobilisation is by altering the physicochemical properties of the metal-soil complex by introducing a multipurpose anion, such as phosphate, that enhances metal adsorption via anion-induced negative charge and metal precipitation (Bolan et al., 2003). Addition of humified organic matter (O.M.) such as compost, together with lime to raise soil pH (Kuo et al., 1985), is a common practice for immobilizing heavy metals and improving soil conditions, to facilitate re-vegetation of contaminated soils (Williamson and Johnson, 1981). Soil acidification, due to the oxidation of metallic sulphides in the soil, increases metal bioavailability; but liming can control soil acidification; and organic materials generally promote fixation of heavy metals in non-available soil fractions, with Cu bioavailability being particularly affected by organic treatments (Clemente et al., 2003). The production of sulphate 21  by sulphide oxidation increases solubility of Zn and Mn, and therefore their concentrations in plant-available (DTPA-extractable) fractions. However, the bioavailability of Cu did not decrease with either soil pH increase or with lime, indicating that the organic treatments might have had a significant effect. Revegetation of mine tailings usually requires amendments of phosphorus, even though phosphate addition can mobilize arsenic (As) from the tailings. Recent research results on phytostabilization are summarized in Table 2.4. Table 2.4. Summary of research results - Phytostabilisation  Plant species  Metal  Treatments  Results  Limitations  Reference  Hordeum vulgare, Lupinus angustifolius, Secale cereale  As  Different P amendment products (organic and inorganic)  P amendment of < 3 g m-2 caused As leaching of 0.5 mg L-1 from unplanted lysimeters and up to 0.9 mg L-1 on average in planted lysimeters. Arsenic accumulated in plant biomass to 126 mg/kg in shoots and 469 mg/kg in roots.  Variable speciesamendment combinations produced differences in the amount of As leached and uptake.  Mains et al., 2006a,b  Lolium italicum and Festuca arundinaceae  Pb and Zn  Compost at two rates (10%, and 30% v/v)  The concentration of Pb and Zn in aerial parts and in roots of L. italicum and F. arundinacea decreased more than five times in presence of compost. Pb content decreased from 218 to 32 mg/kg in shoot and 7232 to 1196 mg/kg in root. Zn decreased from 4190 to 624 mg/kg in shoot and 7120 to 1993 mg/kg in root.  The level of contaminants in aerial parts of plants was still too high to be grazed by herbivores.  Rizzi et al., 2004  Brassica juncea  Cd  Soil amendmentsliming materials, phosphate compounds and biosolids  Phosphate immobilized Cd, thereby reducing the phytotoxicity of Cd. The tissue metal concentration of Cd, Cu and Cr(VI) with biosolids application was 253, 157 and 12.4 mg/kg. (i.e. a decrease over nil amendment).  Bolan et al., 2003  22  Plant species  Metal  Treatments  Results  Limitations  Reference  Brassica juncea  Zn, Cu, Mn, Fe, Pb and Cd  organic amendments (cow manure and compost) and lime  Active phytoremediation followed by natural attenuation, was effective for remediation of pyritepolluted soil. Bioavaialble soil concentration decreased from: 363 to 166 mg/kg for Zn, 36 to 31 mg/kg for Cu, 1.94 to 1.48 mg/kg for Pb, 1.6 to 0.86 mg/kg for Cd, 679 to 303 mg/kg for Fe and 245 to 120 mg/kg for Mn. Available As concentration in soil decreased from 2.5-13.5 mg/kg after the first crop to 0.5-2.6 mg/kg after the second.  Bioavailability of Cu did not decrease with either soil pH increase or with lime.  Clemente et al., 2003, Clemente et al., 2006  Hyparrhenia hirta and Zygophyllum fabago  Pb, Zn and Cu  Characterizat ion of soil and plant samples from a mine tailing located in South-East Spain for further phytostabilisa tion research  H. hirta accumulated around 150 mg kg−1 Pb in both shoots and roots. Zn concentration was 750 mg kg−1 in Z. fabago shoots.  The plant Conesa et species, H. hirta al., 2006 and Z. fabago, colonize only parts of the tailings with low electrical conductivity  Leachates and uptakes of As were found to be higher with an organic fertilizer amendment than super-phosphate, particularly in combination with barley (Mains et al., 2006b). Active phytoremediation followed by natural attenuation, was effective for remediation of the pyritepolluted soil (Clemente et al., 2006). The Met PAD IM bio test was used to assess the extent of metal accumulation by plants in mining areas. Plants were identified as hyper tolerant which can be used for phytostabilization (Boularbah et al., 2006). Two plant species, Hyparrhenia hirta and Zygophyllum fabago, that have naturally colonized some parts of mine tailings in South-East Spain, have been reported to tolerate high metal concentrations in their rhizospheres. These plant species do not take up high  23  concentrations of metals, providing a good tool to achieve surface stabilization of tailings with low risk of affecting the food chain (Conesa et al., 2006). Phytostabilization efforts in the Mediterranean region have been found to be improved by using mixtures including local metallicolous legume and grass species (Fre´rot et al., 2006). It is better to identify the plants spontaneously colonizing the contaminated site, since they are more ecologically adapted than introduced species.  2.2.2 Phytofiltration Phytofiltration is the use of plant roots (rhizofiltration) or seedlings (blastofiltration) to absorb or adsorb pollutants, mainly metals, from water and aqueous-waste streams (Prasad and Freitas, 2003). Plant roots or seedlings grown in aerated water absorb, precipitate and concentrate toxic metals from polluted effluents (Dushenkov and Kapulnik, 2000; Elless et al., 2005). Mechanisms involved in biosorption include chemisorption, complexation, ion exchange, micro precipitation, hydroxide condensation onto the biosurface, and surface adsorption (Gardea-Torresdey et al., 2004). Rhizofiltration uses terrestrial plants instead of aquatic plants because the former feature much larger fibrous root systems covered with root hairs with extremely large surface areas. Metal pollutants in industrial-process water and in groundwater are most commonly removed by precipitation or flocculation, followed by sedimentation and disposal of the resulting sludge (Ensley, 2000). The process involves raising plants hydroponically and transplanting them into metal-polluted waters where plants absorb and concentrate the metals in their roots and shoots (Dushenkov et al., 1995; Salt et al., 1995; Flathman and Lanza, 1998; Zhu et al., 1999). Root exudates and changes in rhizosphere pH may also cause metals to precipitate onto root surfaces. As they become saturated with the metal contaminants, roots or whole plants are harvested for disposal (Flathman and Lanza, 1998; Zhu et al., 1999). Dushenkov et al. (1995), Salt et al. (1995), and Flathman and Lanza (1998) contend that plants for phytofiltration should accumulate metals only in the roots. Dushenkov et al. (1995) explain that the translocation of metals to shoots would decrease the efficiency of rhizofiltration by increasing the 24  amount of contaminated plant residue needing disposal. However, Zhu et al. (1999) suggest that the efficiency of the process can be increased by using plants with a heightened ability to absorb and translocate metals.  Several aquatic species have the ability to remove heavy metals from water, including water hyacinth (Eichhornia crassipes., Kay et al., 1984; Zhu et al., 1999), pennywort (Hydrocotyle umbellata L., Dierberg et al., 1987), and duckweed (Lemna minor L., Mo et al., 1989). However, these plants have limited potential for rhizofiltration because they are not efficient in removing metals as a result of their small, slow-growing roots (Dushenkov et al., 1995). The high water content of aquatic plants complicates their drying, composting, or incineration. In spite of limitations, Zhu et al. (1999) indicated that water hyacinth is effective in removing trace elements in waste streams. Sunflower (Helianthus annus L.) and Indian mustard (Brassica juncea Czern.) are the most promising terrestrial candidates for removing metals from water. The roots of Indian mustard are effective in capturing Cd, Cr, Cu, Ni, Pb, and Zn (Dushenkov et al., 1995), whereas sunflower removes Pb (Dushenkov et al., 1995), U (Dushenkov et al., 1997a),  137  Cs, and  90  Sr (Dushenkov et al., 1997b) from hydroponic  solutions. A novel phytofiltration technology has been proposed by Sekhar et al. (2004) for removal and recovery of lead (Pb) from wastewaters. This technology uses plant-based biomaterial from the bark of the plant commonly called Indian sarsaparilla (Hemidesmus indicus). The target of their research was polluted surface water and groundwater at industrially contaminated sites. Cassava waste biomass was also effective in removing two divalent metal ions, Cd (II) and Zn (II), from aqueous solutions (Horsfall and Abia, 2003). Modification of the cassava waste biomass by treating it with thioglycollic acid resulted in increased adsorption rates for Cd, Cu, and Zn (Abia et al., 2003). Several species of Sargassum biomass (non living brown algae) were effective biosorbents for heavy metals such as Cd and Cu (Davis, et al., 2000).  25  Table 2.5. Summary of research results - Phytofiltration Plant species  Metal  Treatments  Brassica juncea, Helianthus annuus  Cu, Cd, Roots of Cr, Ni, hydroponically Pb, and grown terrestrial Zn plants used to remove toxic elements from aqueous solutions  Sunflower  U  Water Hyacinth  As, Cd The abilities of Cr, Cu, water hyacinth to Ni, and take up and Se translocate six trace elements-As, Cd, Cr, Cu, Ni, and Se were studied under controlled conditions  Duckweed  Hg  Duckweed (Lemna minor L.) and water velvet (Azolla pinnata).  Fe and Solutions enriched Cu with 1·0, 2·0, 4·0, and 8·0 ppm of these 2 metal ions, renewed every 2 days over a 14-day test period.  Results  Reference  Roots of B. juncea concentrated Dushenkov these metals 131-563-fold (on a et al., 1995 DW basis) above initial solution concentrations. The recoveries of heavy metals were 45 % for Cd, 55% for Zn, 50% for Cr, 45% for Ni, 97% for Cu and 100 % for Pb.  Rhizofiltration of U concentration in water Dushenkov U in water by reduced from 21-874 ug/l to et al., 1997 roots of sunflower <20 ug/l by rhizofiltration plants The highest levels of Cd in Zhu et al., shoots and roots were 371 and 1999 6103 mg/kg dry wt., and those of Cr were 119 and 3951 mg/kg dry wt., Cadmium, Cr, Cu, Ni, and As were more highly accumulated in roots, whereas Se accumulated more in shoots.  Effects of pH, Duckweed strongly absorbed Mo et al., copper and humic Hg from water and after 3 days 1989 acid contained 2000 ppm of Hg by weight When duckweed was kept in a Jain et al., solution containing Cu alone at 1989 8·0 ppm level, the value of the metal concentration factor after 14 days was 51.20. However, in the presence of an equal concentration of Fe the value of this factor was 26.53, indicating the influence of Fe on the uptake rate of Cu.  26  Plants used for phytofiltration should be able to accumulate and tolerate significant amounts of the target metals, in conjunction with easy handling, low maintenance costs, and a minimum of secondary waste requiring disposal. It is also desirable for plants to produce significant amounts of root biomass or root surface area (Dushenkov and Kapulnik, 2000). Reports on phytofiltration are summarized in Table 2.5.  2.2.3 Phytovolatilization Some metal contaminants such as As, Hg, and Se may exist as gaseous species in the environment. In recent years, researchers have sought naturally-occurring or geneticallymodified plants capable of absorbing elemental forms of these metals from the soil, biologically converting them to gaseous species within the plant, and releasing them into the atmosphere. This process is called phytovolatilization. The mechanism of phytovolatilization is shown schematically in Figure 2.4.  Figure 2.4  Schematic mechanism of phytovolatilization.  Volatilization of Se from plant tissues may provide a mechanism of selenium detoxification. As early as 1894, Hofmeister proposed that selenium in animals is detoxified by releasing volatile dimethyl selenide from the lungs, based on the fact that the odour of dimethyl telluride was detected in the breath of dogs injected with sodium tellurite. Using the same logic, it was suggested that the garlicky odour of plants that accumulate selenium may indicate release of 27  volatile selenium compounds. This is the most controversial of phytoremediation technologies. Hg and Se are toxic (Suszcynsky and Shann, 1995), and there is doubt about whether the volatilization of these elements into the atmosphere is desirable or safe (Watanabe, 1997). The volatile selenium compound released from the selenium accumulator Astragalus racemosus was identified as dimethyl diselenide (Evans et al., 1968). Selenium released from alfalfa, a selenium nonaccumulator, was different from the accumulator species and was identified as dimethyl selenide. Lewis et al. (1966) showed that both selenium nonaccumulator and accumulator species volatilize selenium. Selenium phytovolatilization has received the most attention to date (Lewis et al., 1966; Terry et al., 1992; Bañuelos et al., 1993; McGrath, 1998) because this element is a serious problem in many parts of the world where there are Se-rich soils (Brooks, 1998). According to Brooks (1998), the release of volatile Se compounds from higher plants was first reported by Lewis et al. (1966). Terry et al. (1992) report that members of the Brassicaceae are capable of releasing up to 40 g Se ha-1 day  -1  as various gaseous  compounds. Some aquatic plants, such as cattail (Typha latifolia L.), have potential for Se phytoremediation (Pilon-Smits et al., 1999). Volatile Se compounds such as dimethyl selenide are 1/600 to 1/500 as toxic as inorganic forms of Se found in soil (DeSouza et al., 2000). The volatilization of Se and Hg is also a permanent site solution, because the inorganic forms of these elements are removed, and gaseous species are not likely to redeposit at or near the site (Atkinson et al., 1990; Heaton et al., 1998). Furthermore, sites that utilize this technique may not require much management after the original planting. This remediation method has the added benefits of minimal site disturbance, less erosion, and no need to dispose of contaminated plant material (Heaton et al., 1998). Heaton et al. (1998) suggest that the transfer of Hg (O) to the atmosphere would not contribute significantly to the atmospheric pool. This technique appears to be a promising tool for remediating Se- and Hg- contaminated soils. Volatilization of arsenic as dimethyl arsenite has also been postulated as a resistance mechanism in marine algae. However, it is not known whether terrestrial plants also volatilize arsenic in significant quantities. Studies on arsenic uptake and distribution in higher plants indicate that arsenic predominantly accumulates in roots and that only small quantities are transported to  28  shoots. However, plants may enhance the biotransformation of arsenic by rhizospheric bacteria, thus increasing the rates of volatilization (Salt et al., 1998). Unlike other remediation techniques, once contaminants have been removed via volatilization, there is a loss of control over their migration to other areas. Some authors suggest that the addition to atmospheric levels through phytovolatilization would not contribute significantly to the atmospheric pool, since the contaminants are likely to be subject to more effective or rapid natural degradation processes such as photodegradation (Azaizeh et al., 1997). However, phytovolatilization should be avoided for sites near population centres and at places with unique meteorological conditions that promote the rapid deposition of volatile compounds (Heaton et al., 1998).  Hence the consequences of releasing the metals to the atmosphere need to be  considered carefully before adopting this method as a remediation tool.  2.2.4  Phytoextraction  Phytoextraction, also called phytoaccumulation, refers to the uptake and translocation of metal contaminants in the soil by plant roots into above-ground components of the plants (Figure 2.5). The typical levels of metal concentration effects in plants are given in Table 2.6.  Figure 2.5  Schematic mechanism of phytoextraction. 29  Table 2.6 Effect of typical levels for metals in plants  Status  Metal concentrations (mg/kg) Cd  Cu  Pb  Zn  Deficient  –  <1–5  –  <10  Normal  0.05–2  3–30  0.5–10  10–150  Phytotoxic  5–700  20–100  30–300  >100  Adapted from Pugh et al. (2002)  Table 2.7(a). Examples of hyperaccumulators and their bioaccumulation potential.  Plant species  Metal  Content (mg/kg)  Reference  Thlaspi caerulescens  Zn  39,600 (shoots)  Reeves and Brooks (1983)  Thlaspi caerulescens  Cd  1800  Baker and Walker (1990)  Ipomea alpine  Cu  12300  Baker and Walker (1990)  Sebertia acuminate  Ni  25% by wt. dried sap  Haumaniastrum robertii  Co  10,200  Brooks (1998)  Astragalus racemosus  Se  14 900  Beath et al. (1937)  Pteris vittata  As  27,000  Wang et al, 2002  Berkheya coddii  Ni  5500  Robinson et al., 1997  Iberis intermedia  Ti  3070  Leblanc et al., 1999  Jaffre et al. (1976)  30  Table 2.7 (b) Hyperaccumulators and their bioaccumulation potential. Results  Plant species  Metal  Pistia stratiotes  Ag, Cd, Cr, All elements accumulated mainly in the Cu, Hg, Ni, root system. Pb and Zn. Hg Organic Hg was absorbed and transformed into an inorganic form (Hg+, Hg2+) and accumulated in roots. Pb Pb concentrated in the leaf and stem indicating the prerequisites of a hyperaccumulator plant. Pb Heavy metal mainly accumulated in roots and shoots. Pb Pb accumulated as lead acetate in roots and leaves, although lead sulfate and sulfide were also detected in leaves, whereas lead sulfide was detected in root samples. Lead nitrate in the nutrient solution biotransformed to lead acetate and sulfate in its tissues. Complexation with acetate and sulfate may be a lead detoxification strategy in this plant species. As A preliminary bioindicator for As transfer from substrate to plants. Used for As phytoremediation of mine tailing waters because of its high accumulation capacity. As Suitable for phytoremediation in the moderately contaminated soils.  Spartina plants  Helianthus annuus Hemidesmus indicus Sesbania drummondii  Lemna gibba  Pteris vittata, P. cretica, P. longifolia and P. umbrosa. Alyssum  Solanum nigrum and Conyza canadensis  Ni  Cd  Reference Odjegba and Fasidi, 2004 Tian et al., 2004  Boonyapookana et al., 2005 Chandra et al., 2005 Sharma et al., 2004  Mkandawire and Dudel, 2005  Caille et al., 2004  Majority of Ni is stored either in the leaf Broadhurst et al., epidermal cell vacuoles, or in the basal 2004 portions of the numerous stellate trichomes. The metal concentration in the trichome basal compartment was the highest ever reported for healthy vascular plant tissue, approximately 15-20% dry weight. High concentration of Cd accumulated. Wei et al., 2004 Tolerant to combined action of Cd, Pb, Cu and Zn 31  Plant species  Metal  Results  Reference  Thlaspi caerulescens  Cd  High Cd-accumulating capability, Schwartz et al., acquiring Cd from the same soil pools 2003 as non-accumulating species.  Arabis gemmifera  Cd and Zn  Hyperaccumulator of Cd and Zn, with Kubota and phytoextraction capacities almost equal Takenaka, 2003 to Thlaspi caerulescens.  Stanleya pinnata  Se  Adapted to semi-arid western U. S. soils Parker et al., and environments. Uptake, metabolism 2003 and volatilization of Se.  Austromyrtus bidwilli . Phytolacca acinosa Roxb  Mn  Australian native hyperaccumulator of Bidwell et al., Mn, grows rapidly, has substantial 2002, Xue et al., biomass, wide distribution and a broad 2004 ecological amplitude  The terms phytoremediation and phytoextraction are sometimes incorrectly used as synonyms, but phytoremediation is a concept, whereas phytoextraction is a specific clean-up technology (Prasad and Freitas, 2003). Certain plants, called hyperaccumulators, absorb unusually large amounts of metals compared to other plants and the ambient metals concentration. Natural metal hyperaccumulators are plants that can accumulate and tolerate greater metal concentrations in shoots than those usually found in non-accumulators, without visible symptoms. Examples of commonly reported hyperaccumulators are given in Tables 2.7(a) and (b). According to Baker and Brooks (1989), hyperaccumulators should have a metal accumulation exceeding a threshold value of shoot metal concentration of 1% (Zn, Mn), 0.1% (Ni, Co, Cr, Cu, Pb and Al), 0.01% (Cd and Se) or 0.001% (Hg) of the dry weight shoot biomass. Over 400 hyperaccumulator plants have been reported, including members of the Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae, Cunouniaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, Violaceae, and Euphobiaceae. Recently Environment Canada has released a database "Phytorem" which contains a worldwide inventory of more than 750 terrestrial and aquatic plants, both wild and cultivated species and varieties, of potential value for phytoremediation. These plants are selected and planted at a site based on the metals present and site conditions. After they have grown for several weeks or months, the plants are harvested. Planting and harvesting may be repeated to reduce contaminant levels to allowable limits (Kumar et al., 1995). The time required for remediation depends on the type and extent of metal contamination, 32  the duration of the growing season, and the efficiency of metal removal by plants, but it normally ranges from 1 to 20 years (Kumar et al., 1995; Blaylock and Huang, 2000). This technique is suitable for remediating large areas of land contaminated at shallow depths with low to moderate levels of metal-contaminants (Kumar et al., 1995; Blaylock and Huang, 2000).  2.2.4.1 Types of phytoextraction Two basic strategies of phytoextraction are being developed: chelate-assisted phytoextraction, which we term induced phytoextraction; and long-term continuous phytoextraction. If metal availability is not adequate for sufficient plant uptake, chelates or acidifying agents may be added to the soil to liberate them (Cunningham and Ow, 1996; Huang et al., 1997; Lasat et al., 1998). However, side effects of the addition of chelate to the soil microbial community are usually neglected. It has been reported (Wu et al., 1999) that many synthetic chelators capable of inducing phytoextraction might form chemically and microbiologically stable complexes with metals, threatening soil quality and groundwater contamination. Several chelating agents, such as EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-O,O'-bis-[2-amino-ethyl]N,N,N',N',-tetra acetic acid), EDDHA (ethylenediamine di o-hyroxyphenylacetic acid), EDDS (ethylene diamine disuccinate) and citric acid, have been found to enhance phytoextraction by mobilizing metals and increasing metal accumulation (Tandy et al., 2006; Cooper et al., 1999). The increase in the phytoextraction of Pb by shoots of Zea mays L. was more pronounced than the increase of Pb in the soil solution with combined application of EDTA and EDDS (Luo et al., 2006). Although EDTA was, in general, more effective in soil metal solubilization, EDDS, less harmful to the environment, was more efficient in inducing metal accumulation in Brachiaria decumbens shoots (Santos, et al., 2006). However, there is a potential risk of leaching of metals to groundwater, and a lack of reported detailed studies regarding the persistence of metal-chelating agent complexes in contaminated soils (Lombi et al., 2001a,b).  2.2.4.2 Successful factors for phytoextraction of heavy metals As a plant-based technology, the success of phytoextraction is inherently dependent on several plant characteristics, the two most important being the ability to accumulate large quantities of biomass rapidly and the capacity to accumulate large quantities of environmentally important metals in the shoot tissue (Kumar et al., 1995; Cunningham and Ow, 1996; McGrath, 1998, 33  Pilon-Smits, 2005). Effective phytoextraction requires both plant genetic ability and the development of optimal agronomic practices, including (i) soil management practices to improve the efficiency of phytoextraction, and (ii) crop management practices to develop a commercial cropping system. Ebbs et al. (1997) reported that B. juncea, while having one-third the concentration of Zn in its tissue, is more effective at removing Zn from soil than Thlaspi caerulescens, a known hyperaccumulator of Zn. The advantage is due primarily to the fact that Brassica juncea produces ten-times more biomass than Thlaspi caerulescens. Plants for phytoextraction should be able to grow outside their area of collection, have profuse root systems and be able to transport metals to their shoots. They should have high metal tolerance, be able to accumulate several metals in large amounts, exhibit high biomass production and fast growth, resist diseases and pests, and be unattractive to animals, minimizing the risk of transferring metals to higher trophic levels of the terrestrial food chain (Thangavel and Subhuram, 2004). Phytoextraction is applicable only to sites containing low to moderate levels of metal pollution, because plant growth is not sustained in heavily polluted soils. The land should be relatively free of obstacles, such as fallen trees or boulders, and have an acceptable topography to allow normal cultivation practices, utilizing agricultural equipment. Selected plants should be easy to establish and care for, grow quickly, have dense canopies and root systems, and be tolerant of metal contaminants and other site conditions which may limit plant growth. Basic et al. (2006a,b) investigated the parameters influencing the Cd concentration in plants, as well as the biological implications of Cd hyperaccumulation in nine natural populations of Thlaspi caerulescens. Cd concentrations in the plant were positively correlated with plant Zn, Fe and Cu concentrations. The physiological and/or molecular mechanisms for uptake, transport and/or accumulation of these four metals interact with each other. They specified a measure of Cd hyperaccumulation capacity by populations and showed that Thlaspi caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth, by developing more and bigger leaves, taller stems, and produced more fruits and heavier seeds. Liu et al. (2006) conducted a survey of Mn mine tailing soils and eight plants growing on Mn mine tailings. The concentrations of soil Mn, Pb, and Cd and the metal-enrichment traits of these eight plants were analyzed. It was found that Poa pratensis, Gnaphalium affine, Pteris vittata, Conyza canadensis and Phytolacca acinosa possessed specially good metal-enrichment and metal-  34  tolerant traits. In spite of the high concentration of Mn in P. pratensis, its lifecycle was too short, and its shoots were too difficult to collect for it to be suitable for soil remediation. The effectiveness of phytoextraction of heavy metals in soils also depends on the availability of metals for plant uptake (Li et al., 2000). The rates of redistribution of metals and their binding intensity are affected by the metal species, loading levels, aging and soil properties (Han et al., 2003). Generally, the solubility of metal fractions is in the order: exchangeable > carbonate specifically adsorbed > Fe-Mn oxide > organic > residual (Li and Thornton, 2001). Ammonium nutrition of higher plants results in rhizosphere acidification due to proton excretion by root cells. Ammonium-fed sunflowers induced a strong acidification of the solution and, compared to the nitrate-fed sunflowers, a small modification in mineral nutrition and different Cd partitioning between root and shoot. Moreover, ammonium nutrition was found to induce a great mobilisation of a sparingly soluble form of cadmium (CdCO3) (Zaccheo et al., 2006). A lipidtransfer protein isolated from a domestic cultivar of barley grain, Hordeum vulgare has the affinity to bind Co (II) and Pb (II), but not Cd (II), Cu (II), Zn (II) or Cr (III). This suggests a new possible role of barley lipid-transfer protein for phytoextraction (Gorjanovic et al., 2006). The slow desorption of metals in soils has been a major impediment to the successful phytoextraction of metal contaminated sites. Except for Hg, metal uptake into roots occurs from the aqueous phase. In soil, easily mobile metals such as Zn and Cd occur primarily as soluble or exchangeable, readily bioavailable form. Cu mainly occurs as organic complexes and Mo predominate in inorganically bound and exchangeable fractions. Slightly mobile metals such as Ni and Cr are mainly bound in silicates (residual fraction). Soluble, exchangeable and chelated species of trace elements are the most mobile components in soils, facilitating their migration and phytoavailability (Williams et al., 2006). Other species such as Pb occur as insoluble precipitates (phosphates, carbonates and hydroxyl-oxides) which are largely unavailable for plant uptake (Pitchel et al., 1999). Understanding the mechanisms of rhizosphere interaction, uptake, transport and sequestration of metals in hyperaccumulator plants will lead to designing novel transgenic plants with improved remediation traits (Eapen and Souza, 2005). Moreover, the selection and testing of multiple hyperaccumulator plants could enhance the rate of phytoremediation, giving this process a promise one for bioremediation of environmental contamination (Suresh and Ravishankar, 2004). Some of the recent reports on phytoextraction are summarized in Table 2.8. 35  Table 2.8. Recent reports on phytoextraction  Metal Plant studied  Method of Results Phytoremediation  Reference  Cd, Zn  Thlaspi caerulescens  PE-C  Physiological and molecular mechanisms for uptake, transport and accumulation of four heavy metals Cd, Fe, Cu and Zn interact with each other. T. caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth. Revegetation of metal polluted soils with T. caerulescens could help activate their biochemical and microbial functionality. Different soils had various responses to acidification. A different optimum pH may exist for phytoextraction.  Basic et al., 2006a; Basic et al., 2006b; Keller et al.,2006; Hammer et al.,2006; HernandezAllica et al. (2006);.Wang et al. (2006)  Mn  Gnaphalium affine D. Don Conyza canadensis (L.) Cronq  PE-C  G. affine and C. canadensis had Liu et al, excessive accumulation of Mn and 2006; Xue et could be useful in al., 2004 phytoremediation. The perennial herb Phytolacca acinosa Roxb. (Phytolaccaceae), which occurs in Southern China, was found to be a new manganese hyperaccumulator.  Cu  Elsholtzia splendens, and Trifolium repens  PE-CA  Application of glucose or citric Chen et al., acid significantly increased the 2006 extractable Cu concentration in planted and unplanted soils. Concentrations of Cu in the shoots of E. splendens were 2.6, 1.9 and 2.9 times of those of T. repens under no chelate, citric acid and glucose treatments, respectively.  Pb, As,  Carrot, Lettuce and Tomato.  PE-C  Pb, Cu, Zn, Cd.  Euphorbia,Verbascum and Astragalus.  Except for carrot roots concentration less than ICP-OES detectable limits. Plants with high metal intake abilities escalate mobility of metals and increase contaminations on surface and subsurface.  Pendergrass and Butcher (2006). Sagiroglu et al. (2006)  36  Metal Plant studied  Method of Results Phytoremediation  Reference  Cu, Zn, Pb  Sunflower  PE-CA  Synthetic chelating agents did not increase the uptake of metals for equal soluble concentrations in the presence and absence of chelates. Proper use of soil amendments increased the phytoextraction of Zn, Cu, Pb, Cd from contaminated soils.  Tandy et al., 2006; Clemente et al., 2006; Chen et al., 2006  Cu and Fe  Athyrium vokoscense  PE and PM  1 g Cu and 0.1 g Fe recovered from 500 g soil. Removal rates of Cu and Fe in the contaminated soil were 82 and 95 % respectively. Application of (NLMWOA (Natural Low Molecular Weight Organic Acids) increased the extraction of Cu, with no enhancement of lead phytoextraction.  Kobayashi et al., 2006; Evangelou et al., 2006  Se  Astragalus bisulcatus and Brassica juncea  PE  There was a substantial LeDuc et al., improvement in Se accumulation 2006 (4 to 9 times increase) in transgenic plants.  Cd  Brassica napus and Brassica juncea  PE  Lipid changes in B. juncea, the well- known Cd-hyperaccumulator species, revealed greater stability of its cellular membranes to cadmium-stress compared to a Cdsensitive specie, B. napus.  Quartacci et al., 2006; Belimov et al., 2005; Nouairi et al., 2006; Sheng and An increase in cadmium content Xia, 2006 varying from 16 to 74%, compared to the non-inoculated control, was observed in rape plants cultivated in soil treated with 100 mg Cd kg−1 (as CdCl2) and inoculated with the cadmium-resistance bacterial strains from heavy metalpolluted soils.  PE- PhytoExtraction, CA- Chelate Assisted, C- Continuous, PM- Phytomining  37  Phytoremediation has been combined with electrokinetic remediation, applying a constant voltage of 30 V across the soil. The combination of both techniques could represent a very promising approach to the decontamination of metal-polluted soils (O'Connor et al., 2003).  2.3 Handling of hazardous plant biomass after phytoremediation Phytoextraction involves repeated cropping of plants in contaminated soil until the metal concentration drops to an acceptable level. Each crop is removed from the site. This leads to accumulation of huge quantities of hazardous biomass, which must be stored or disposed appropriately to minimize environmental risk. After harvesting, the methods of disposal of contaminated plants include approved secure landfills, surface impoundments, deep well injection, ocean dumping or incineration. The waste volume can be reduced by thermal, microbial, physical or chemical means. In one study, the dry weight of Brassica juncea for induced phytoextraction of lead amounted to 6 tons/ha containing 10,000 to 15,000 mg/kg metal on a dry weight basis (Blaylock et al., 1997). Composting and compaction can provide postharvest treatment (Raskin et al., 1997 and Kumar et al., 1995). Even though composting can significantly reduce the volume of the harvested biomass, metalcontaminated biomass still requires treatment prior to disposal. In the case of compaction, care should be taken to collect and dispose of the leachate. A conventional and promising route to utilize biomass produced by phytoremediation is through thermo-chemical conversion processes such as combustion, gasification and pyrolysis. If phytoextraction could be combined with biomass generation and its commercial utilization as an energy source, then it could be turned into a profitable operation, with the residual ash available to be used as an ore (Brooks, 1998; Comis, 1996; Cunningham and Ow, 1996). Phytomining includes the generation of revenue by extracting soluble metals produced by the plant biomass ash, also known as bio-ore. With some metals like Ni, Zn, Cu, etc., the value of reclaimed metal may provide an additional incentive for phytoremediation (Chaney et al.1997, Watanabe 1997, Thangavel and Subhuram 2004).  38  2.4. Conclusions Phytoremediation is still in its research and development phase, with many technical issues needing to be addressed. The results, though encouraging, suggest that further development is needed. Phytoremediation is an interdisciplinary technology that can benefit from many different approaches. Results already obtained have indicated that some plants can be effective in toxic metal remediation. The processes that affect metal availability, metal uptake, translocation, chelation, degradation, and volatilization need to be investigated in detail. Better knowledge of these biochemical mechanisms may lead to: (1) Identification of novel genes and the subsequent development of transgenic plants with superior remediation capacities; (2) Better understanding of the ecological interactions involved (e.g. plant–microbe interactions); (3) Appreciation of the effect of the remediation process on ecological interactions; and (4) Knowledge of the entry and movement of the pollutant in the ecosystem. In addition to being desirable from a fundamental biological perspective, findings will help improve risk assessment during the design of remediation plans, as well as alleviation of risks associated with the remediation. It is important that public awareness of this technology be considered, with clear and precise information made available to the general public to enhance its acceptability as a global sustainable technology. 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Depending on the strategy adopted by plants, the remediation method can be either containment or removal (Thangavel and Subhuram, 2004). Containment by stabilisation may be suited for busy contaminated sites like highway soils where removal is neither feasible nor practical due to financial and other physical constraints. An evaluation of contaminant metal status and phytoremediation potential of local plants of the site is essential prior to an in situ soil phytoremediation program. Field measurements of metal accumulation (Cu, Pb, Mn and Zn) in soils and plants along highway sites and the potential for phytostabilisation of plant species were investigated. The selected sites for the present study (Appendix A, Figures 1 and 2) are located on the northern right of way of the Trans-Canada Highway (TCH) or HW1 close to the intersection with the 176th Street overpass in Surrey and northern ramp of HW 17, Deltaport Way, Delta, British Columbia. Both highway sites have similar design i.e. elevated highway sections with overflow flush shoulder type of drainage, but with different surrounding geology, land use, average daily traffic, and predominant meteorological conditions. The overall objectives of this study were to charecterise the physico-chemical properties of highway soils, to characterise the extent and type of metal contamination (Pb, Zn, Cu and Mn) in these soils, to identify the plants that spontaneously colonise the polluted sites and to assess their metal accumulation capacities.  2  A version of this chapter has been published. Padmavathiamma, P.K, Li, L.Y. and Lavkulich, L. (2007) Heavy metal contamination and potential of local plants for phytoremediation along Highways. Biogeochemistry of Trace Elements: Environmental Protection, Remediation and Human Health, Edited: Y. Ahu, N. Lepp and R Naidu. pp.648-650. 53  Information obtained from this study provided insight for formulating the technical programs for subsequent studies to identify a package for remediating the metal-contaminated sites.  3.2 Materials and methods  3.2.1 Site description th  The study was focused on two highway sites, Trans-Canada Highway (HW 1) at the 176 Street intersection in Surrey, B.C and Highway 17 in Delta, B.C. Factors considered in site selection include: traffic characteristics, surrounding land use activities, meteorological conditions, pavement type and condition, drainage area, highway design characteristics, proximity to a receiving water body, highway maintenance practices, and logistical considerations (safety, accessibility, future development, etc.). For this study, both sites share most characteristics (except for traffic numbers, land use and meteorological conditions) and are thus similar enough to be suitable for comparison, but different enough to test the efficiency of the selected methodology. Having two different study sites served two purposes: 1) to observe the influence of different geo-environmental conditions on soil metal loadings and plant accumulations, and 2) to assess the efficiency of the selected methodology for remediation in soils from two different road-side conditions. th  3.2.1.1 Trans-Canada Highway & 176 St. site description This site is located on the northern right of way of the Trans-Canada Highway (TCH), close to th  the intersection with the 176 Street overpass in Surrey, B.C. The highway corridor is oriented in a NW-SE direction and through the Port Mann bridge connects Surrey to Coquitlam and Port Coquitlam. The surface geology is classified as Capilano sediments, which are composed of marine to glaciomarine stony to stoneless silty loam to clayey loam with minor sand and silt. These sediments are normally less than 3 m thick, but in some areas may reach up to 30 m thick (Geological Survey of Canada,1998). Ministry of Transportation and Infrastructure records show traffic counts of 82,900 vehicles per day for the west-bound (WB) corridor and 73,100 for the 54  east-bound (EB). The surface comprises three lanes of asphalt pavement for each direction with a middle grassy area, flush shoulder type of surface drainage and a concrete barrier that separates on and off-ramps from the main corridor in the proximity of the 176  th  street overpass.  Construction of Highway 1 in this location was completed in the 1960s. Surrounding land use is a mixture of residential, agricultural, undeveloped and parkland. Characteristics of this study site are summarized in Appendix A, Table 1.  3.2.1.2 Highway 17 site The study area is located on the northern ramp of HW 17, Deltaport way, Delta, B.C. At the study site location, this highway is oriented in a north-south direction. On a bigger scale Hwy 17 connects Tsawwassen and the ferry terminal to Highway 99, which in turn connects to the rest of the Lower Mainland. The area is located in the flat lowlands of the Fraser River delta. The underlying material is composed of soil deposits, primarily silts, clays and sands. These sediments were deposited over thousands of years by seasonal floodwaters that spread across these lowlands. They are important agricultural soils although in some cases poor drainage can be a problem. The BC Ministry of Environment, Lands & Parks has classified aquifers in the area as having moderate to high vulnerability to contamination, with low to moderate use (Geological Survey of Canada, 1998). The construction of Highway 17 was completed in the early 1970s. Surrounding land use in the area is agricultural. According to Ministry of Transportation and Infrastructure records, traffic counts for HW 17 are 20,417 and 22,899 vehicles/day for Northbound (NB) and Southbound (SB), respectively. The surface comprises two lanes in each direction made of asphalt pavement with flush shoulder surface drainage and a middle concrete barrier. Highway 17 characteristics are summarized in Appendix A, Table 2.  3.2.1.3 Background locations It is important to distinguish between anthropogenic contamination and background or natural levels to enable accurate evaluation of the degree of contamination in an area. The background site for HW 1 is at the bak yard of Surrey fire hall, 1 km north of the main intersection, HW1 with 176 street in Surrey, B.C, whereas the background site for HW 17 is near the Boundary Bay airport, which is north west of the main HW 17 site. The soils in these regions have minimal  55  impacts from anthropogenic effects and have the same physico-chemical characteristics as that of main soils.  3.2.2 Collection of samples and laboratoratory analysis Soil sampling was performed in transects normal to the road. Samples were collected at intervals of 1, 2, 4, 6 and 8 m from the side of the road and at depths of 0-15 and 15-30 cm. Past experiences from Ministry of Transportation and Infrastructure with roadside soils of B.C have shown that metal contamination from above ground sources is generally restricted to the surface layers (Preciado and Li, 2006). The rationale for sampling at different horizontal distances and depths was to assess the influence of traffic behaviour on the migration of metals in the soil. Vigorously growing plants in the study sites were also collected. The soil samples were air-dried for 5 days, and then sieved through a 2-mm mesh after crushing the clods gently using a wooden mallet. Plant samples were thoroughly washed with running tap water and rinsed with de-ionized water to remove soil/sediment particles attached to the plant surfaces. Shoots and roots were then separated and oven dried (70°C) to constant weight. The dried tissues were weighed and finely ground for the analysis of Cu, Pb, Mn and Zn. Basic characteristics of the soil such as pH, electrical conductivity, total carbon, texture, CEC were estimated (Table 3.1). The recoverable heavy metals in the soil sample were estimated by the method proposed by EPA (Smoley, 1992). After dry ashing the plant samples (Lintern et al., 1997), the ash was dissolved in 10 mL of 1 M HCl and diluted to 50 mL with de-ionized water. Both soil and plant extracts were analysed for metals using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Statistical techniques were used to aid in the sampling and interpretation of results. The computer software ORIGIN along with EXCEL was used for statistical computations, including mean values, standard deviation (SD) and metal concentration ratios. To assess the accumulation characteristics and translocation properties of metals in plants, Enrichment Coefficient (EC) and Translocation Factor (TF) were determined. ECroot = [Metal]root/[Metal]soil ECshoot = [Metal]shoot/[Metal]soil TF = [Metal]shoot/[Metal]root  56  3.3 Results and discussion The basic characteristics of two highway soils are given below (Table 3.1). Table 3.1 Basic soil characteristics of Highway soils Soil characteristics  pH in water  HW1 (main site)  HW1 (BG site)  HW 17 (mainsite)  HW 17 (BG site)  5.51  5.42  5.60  5.69  1.10 (dS/m)  0.82 (dS/m)  0.73 (dS/m)  0.64 (dS/m)  2.10 %  1.50%  3.90 %  2.2 %  0.18 %  0.13%  0.30 %  0.19%  19 molc kg-1  15 molc kg-1  22 molc kg-1  20 molc kg-1  10.9 mg/kg  10.4 mg/kg  6.1 mg/kg  10.7 mg/kg  Silty clay loam  Silty clay loam  Humic luvic gleysol.  Humic luvic gleysol.  (Hendershot et al., 2008a) Electrical Conductivity (Lavkulich, 1981) Total Carbon (Sheldrick, 1984) Total Nitrogen Cation Exchange Capacity (Hendershot et al., 2008b) Available P (Bray and Kurtz, 1945) Texture  Sandy clay loam Sandy clay loam  (Kettler et al., 2001) Soil classification  Luvisolic humoferric podzol  Luvisolic humoferric podzol  Concentrations of Cu, Pb, Mn and Zn in soil from two highway sites were found to decrease with increasing distance from the road (Table 3.2 and Figure 3.1). Background (BG) concentrations are given in Table 3.3. The total metal concentrations in HW1 soil were found to be higher than for HW 17 soil, probably because of higher traffic for HW1 compared to HW 17. The decrease of the metal concentrations with distance from the road indicates that vehicular emissions play a significant role in determining the levels of heavy metals in the roadside soil. In the case of Pb, a rapid decline with distance was observed, probably the result of the highly 57  immobile nature of Pb. There have been reports of higher levels of Pb in top soil along major roads in several other cities and of correlations of the metals with traffic volume (e.g. Onianwa, 2001). With respect to Cu and Zn, even though a declining trend was observed with distance from the road, the variation was relatively modest. Typically, abrasions of motor vehicle parts and vehicle emissions, as well as geological parent material, have been demonstrated to be principal sources of these types of metals in road sediments throughout the world (Onianwa, 2001). The use of Tetraethyl lead (TEL) as an antiknock compound for gasoline engines during the early 1970s and subsequent replacement by methyl cyclopentadienyl manganese tricarbonyl (MMT) might have contributed to the high Pb and Mn concentrations in highway soils. Table 3.2 Soil metal concentrations with distance from the highway (HW1). Depth (cm)  0-15  15-30  0-15  Distance from  15-30  0-15  15-30  0-15  15-30  Metal concentrations (mg/kg) Cu  the Highway  Pb  Mn  Zn  (m) 1  74±8.0  47±2  472±28 156±10  266±8.9  341±7.4  129±5.1 95±4.5  2  47±4.6 55±3.6 264±33 162±4.0  374±4.8  394±4.6  94±4.7  88±6.0  4  32±4.6 26±5.6 210±11 103±8.0 278±10.8 301±13.7  85±7.4  70±6.1  6  28±3.8 20±3.0  54±7.0  30±4.6  230±3.2  270±6.0  81±5.3  64±5.0  8  25±3.3 18±3.7  28±8.0  18±2.9  218±3.4  238±6.6  80±5.9  59±6.3  Mean values ± S.D, n = 3. Table 3.3. Metal concentrations in background (BG) sites (0-15 cm) Background (BG) site  Cu (mg/kg)  Pb (mg/kg)  Mn (mg/kg)  Zn (mg/kg)  HW1 (Fire hall soil)  52±5.7  93±11  215±5.9  70±11  31±9  20±6  81±17  50±11  HW17 (near Boundary Bay air port) Mean values ± S.D, n = 3.  58  Soil metal concentrations (mg/kg)  600 500  (a) HW 1 400 300 200 100 0 1  2  4  6  8  Distance from the Highway (m) Cu  Pb  Mn  Zn  Soil metal concentrations (mg/kg)  600  (b) HW 17  500 400 300 200 100 0 1  2  4  6  8  Distance from the Highway (m) Cu  Pb  Mn  Zn  Figure 3.1 Soil metal concentrations with distance from the highway (0-15 cm), a comparison. (a) HW 1 (b) HW 17, (mean values ± SD, n = 3). Plants were collected from the study site at two different times, first late in February (winter) and then towards the end of July (summer), 2006. The plants collected during February were Juncus effusus, Holcus lanatus, Festuca rubra and a moss, Rhytidiadelphus squarrosus. Those collected during the summer were Equisetum arvense, Rumex occidentalis, Plantago lanceolata, Ranunculus occidentalis and Rumex acetosella. All plants, except for weedy species were 59  assessed for metal accumulation and translocation properties. The plants that spontaneously colonised the two sites are given in Appendix A, Figures 7 and 8. Metal concentrations in the plants seen at highway sites during both seasons (winter and summer) are given in Figure 3.2. In the case of Festuca rubra and Holcus lanatus, the concentration of metals in the roots was greater than that of the shoots, revealing a low translocation rate for metals. This indicates low mobility of metals from the roots to the shoots and immobilization of heavy metals in the roots, suggesting an exclusion strategy (Figure 3.2). The metal concentrations in Juncus effusus (both root and shoot) are found to be lower than those of Festuca rubra and Holcus lanatus and there is not a predominant difference between the root and shoot metal concentrations (Figure 3.2),  Plant metal concentrations (mg/kg)  indicating good translocation of metals to the above ground portions of the plant. 250  200  150  100  50  0  Root  Shoot  Root  Cu  Shoot Pb  Juncus effusus  Figure 3.2  Root  Shoot  Root  Mn Festuca rubra  Shoot Zn  Holcus lanatus  Concentration of metals in the plants that spontaneously colonised the study sites (mean values±SD, n = 3).  Since Festuca rubra and Holcus lanatus have a high root EC (Enrichment Coefficient) and low TF (Translocation Factor) with values <1, (Table 3.4), they have the potential for phytostabilization (Kumar et al., 1995). Because Holcus lanatus belongs to the weedy species, Festuca rubra together with other plants selected from Phytorem (Environment Canada, 2003) were tested in the later studies on phytostabilisation, which are explained in subsequent chapters. The moss (Rhytidiadelphus squarrosus) collected from the study site, was found to accumulate substantial quantities of Pb followed by Mn, Zn and Cu (Figure 3.3). Mosses do not have a developed root system and the metal accumulation may be directly from the atmosphere. Hence it can be used as a bio-indicator for atmospheric deposition of metals.  60  Table 3.4 Root/ Shoot ratio, Enrichment Coefficient (EC) and Translocation Factor (TF) Cu  Pb  Mn  Zn  Plant species  R/S ratio  Festuca rubra  0.91.2  1.2  0.98 0.43 0.63 0.47 0.74 0.93 0.87 0.93 1.27 0.85 0.82  Holcus lanatus  0.160.31  1.1  0.94 0.63 0.51 0.39 0.76 0.50 0.43 0.85 1.50 1.60 0.85  ECR ECS  TF  ECR ECS  TF  ECR ECS  TF  ECR ECS  TF  Metal concentration (mg/kg)  Mean values, n = 3. R/S ratio – Root/Shoot ratio, ECR – Enrichment Coefficient, root, ECS Enrichment Coefficient, shoot, TF – Translocation factor.  250 200 150 100 50 0 Cu  Mn  Pb  Zn  M etals  Figure 3.3  Metal concentrations (mg/kg) in moss (Rhytidiadelphus squarrosus), n = 3.  3.4 Conclusions  •  Concentrations of Cu, Pb, Mn and Zn decreased with increasing distance from the highways.  •  High values for metal accumulation in plants from highway soils were observed and variations among plant species were noticed for metal accumulation.  •  Festuca rubra, which spontaneously colonizes the contaminated site, was found to be an ideal candidate for further phytostabilisation studies. The moss collected from the study site, Rhytidiadelphus squarrosus, was found to be a promising indicator for heavy metal accumulation, especially Pb.  •  The basic information obtained from this study provided insight to formulate the technical program for the subsequent studies. 61  3.5 References Bray, R. H. and Kurtz, L.T. (1945). Determination of total, organic and available forms of phosphorus in soils. Soil Sci. 59, 39-45. Chaney, R .L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E .P., Angle, J .S. and Baker, A .J. M. (1997). Phytoremediation of soil metals. Curr. opin. Biotechnol., 8, 279-283. Hendershot, W. H., Lalande, H. and Duquette, M. (2008a). Soil reaction and exchangeable acidity in soil. In: Carter, M. R. and Gregorich, E. G., eds. Soil sampling and methods of analysis, second edition. CRC Press, Taylor &. Francis, Boca Raton. pp. 173-175. Hendershot, W. H., Lalande, H. and Duquette, M. (2008b). Ion exchange and exchangeable cations. In: Carter, M. R. and Gregorich, E. G. eds. Soil sampling and methods of analysis, second edition. CRC Press, Taylor &. Francis, Boca Raton. pp. 203-205. Kettler, T. A., Doran, J. W. and Gilbert, T. L. (2001). Simplified method for particle-size determination to accompany soil-quality analyses, Soil Sci. Am. J. 65, 849-852. Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavymetals from soils. Environ. Sci. Technol., 29(5), 1232-1 238. Lavkulich, L. M. (1981). Methods manual, Pedology Laboratory. Department of Soil Science, University of British Columbia, Vancouver. Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58(1), 114. Onianwa, P. C. (2001). Roadside topsoil concentrations of lead and other heavy metals in Ibadan, Nigeria. Soil Sediment Contam., 10(6), 577 591. Preciado, H. F. and Li, L. Y. 2006. Evaluation of metal loadings and bioavailability in air, water and soil along two Highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108. Sheldrick, B. H. (1984). Total Carbon, LECO induction furnace. Analytical Methods Manual 1984. Research Branch, Agriculture Canada, Ottawa, ON. Sezgin, N., Ozcan, H. K., Demir, G., Nemlioglu,S. and Bayat, C. (2003). Determination of Heavy Metal Concentrations in Street Dusts in IstanbulE-5 Highway. Environment International, 29, 979-985. Smoley,C. K.(1992). Methods for the determination of metals in environmental samples. Environmental Monitoring systems Laboratory, U.S. E.P.A., Cincinnati, Ohio. Thangavel, P. and Subhuram, C. V. (2004). Phytoextraction - Role of hyper accumulators in metal contaminated soils. Proc. Indian Natn. Sci. Acad., B, 70 (1), 109-130.  62  4.  3  PHYTOREMEDIATION OF METAL-CONTAMINATED SOIL IN TEMPERATE  HUMID REGIONS OF BRITISH COLUMBIA, CANADA  4.1 Introduction Metal contamination in soils usually results from industrial activities, such as mining and smelting, electroplating, gas exhaust, energy and fuel production, fertilizer and pesticide application, and municipal waste (Kabata-Pendias and Pendias, 2001). The highway system is a potential source of metal-contaminants to the surrounding environment through natural mechanisms such as atmospheric dust deposition or through the hydrologic cycle (Preciado and Li, 2006). Storm water generated by runoff from roads and highways contains metals in toxic concentrations (Barrett et al., 1998; Characklis and Weisner, 1997; Legret and Pagotto, 1999), due to loading from various sources related to vehicles, road construction materials, and road management (Hallberg et al., 2007). High concentrations of metals, particularly lead, zinc, manganese, iron, and copper, in highway runoff result from the wear of brakes, tires and other vehicle parts, leakage of lubricants, exhaust emission etc. (Preciado and Li, 2006). Because these metals do not degrade naturally, their high concentrations in runoff in both particulate and dissolved forms can result in accumulation in roadside soil at levels that are toxic to organisms in surrounding environments (Sansalone and Buchberger, 1997). Exposure to high levels of these metals has been linked to adverse effects on human health and wildlife. Toxic metals damage DNA, and their carcinogenic effects in animals and humans are probably related to their mutagenic ability (Baudouin et al., 2002). Several techniques have been developed to treat storm water runoff in highways. These include treating storm water runoff with a humic filter media, a pelletized compost medium capable of removing up to 82-98% of the metals (Richman, 1997), using straw coated with sulphide compounds to bind the metals (Robert et al., 2003), or a grassy swale to reduce the concentration of metals in the highway runoff (Barrett et al., 1998).  3  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2009) Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation, 11(6): 575-590. 63  Phytoremediation is an effective technique to remediate metals in soils (Salt et al., 1995; Chaney et al., 1997), since it represents a more sustainable, cost-effective and environmentally-friendly tool for cleaning metal-polluted soils than alternative remediation methods (Baker et al., 1994). Reducing the environmental impact by holding the metal-pollutants at the source location in non-mobile forms so that they do not interfere with the normal life processes of the vegetated cover is phytostabilisation (Smith and Bradshaw, 1979). A typical right of way for roads in Canada is around 30 m, and at least 33% of that land in the right of way is unpaved and can support animal life, where metal contamination needs to be remediated. Phytostabilisation requires least maintenance among different phytoremediation techniques, and it could be a feasible and practical method of remediating in roadside soils along highways and for improving highway runoff drainage. Although many plants have been reported to be effective for phytoremediation of metalcontaminated soil, there is a lack of research addressing the phytoremediation of roadside soils subjected to multi-component metal solutions, like those subjected to continuous atmospheric and highway runoff loadings. Many phytoremediation studies were for a single metal and determine the metal uptake by plant at one stage of growth (Hamlin and Parker, 2006; Weng et al., 2005; Meyers et al., 2008). To the best of our knowledge very little work has been done on the phytoremediation efficiencies of plants in a multi-metal-contaminated scenario. Investigating the effects of uptake pattern, biometric characters and biomass accumulation can contribute to the potential for phytoextraction and phytostabilisation. The present study involved a systematic and comprehensive effort to assess the phytoremediation potential of five plant species, commonly available in regions with temperate maritime climate, for a highway soil in southwest British Columbia. The research protocol involved: (1) estimating the metal uptake by plants at different growth stages; (2) assessing the translocation properties and metal accumulation characteristics, then examining the relationship of bio-metrics and biomass of the plants with metal accumulations; and (3) determining the efficiencies of these plants for phytoextraction and phytostabilisation in soils with multiple metal contaminations. Comprehensive pot tests with randomized experimental design using five plant species and four metals commonly found in highway roadside soils (i.e. Cu, Pb, Mn and Zn) were carried out under outdoor conditions. Detailed analyses were carried out to characterize the 64  chemical and physiological aspects of the plants. The results are intended to contribute to identifying suitable plant species for remediation of metals along highways. This will aid in determining best management practices for phytoremediation of metal contamination in soils, due to traffic activities leading to air deposition and highway runoff.  4.2 Materials and methods The soil used for this research was collected from the yard of Surrey Fire Hall No. 5, located 1 km north of the intersection of TCH (Trans Canada Highway) with the 176 Street overpass in Surrey, British Columbia (a busy site with respect to traffic counts, >80,000 vehicles/day). The sampling site has the same soil as the nearby highway intersection (Luttmerding, 1980). The metal concentrations of the soils studied are given in Table 4.1. The soil at this site had a layer of organic enriched debris, about 5 cm thick, which was removed prior to testing as it would not be typical of a highway soil, and the first 15 cm of soil was collected and brought to the laboratory for the experiments. Table 4.1. Metal concentrations according to British Columbia CSR (Contaminated Sites Regulation) standards and studied soil metal concentrations in the pot study. British Columbia Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) Zn (mg/kg) CSR Standards A 30 50 200 80 B 100 500 1000 500 C 500 1000 2000 1500 Studied Soils B0 52 93 215 70 BA 80 146 408 148 BC 520 1100 2160 1600 Level A is the investigation standard for residential and recreational land use. Level B is the remediation standard for residential and recreational land use, and the investigation standard for commercial and industrial use. Level C is the remediation standard for exclusive commercial and industrial activities (Ministry of Environment, British Columbia, 1995). 4.2.1 Experimental details The concentrations of metals studied were based on previous work (Fakayode and Olu-Owolabi, 2003; Preciado and Li, 2006) and the British Columbia Standards for contaminated sites (Ministry of Environment, British Columbia, 1995). Soils were studied with three different metal 65  concentrations: (a) B0 the original soil containing the following concentrations of the four metals which are the focus of this study: Cu 52 mg/kg, Pb 93 mg/kg, Mn 215 mg/kg, Zn 70 mg/kg. (b) BA the original soil spiked with addition of all four metals to give total concentrations of Cu, Pb, Mn, and Zn of 80, 146, 408 and 148 mg/kg, respectively. (c) BC the original soil spiked to provide total Cu, Pb, Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg, respectively. Table 4.1 summarizes these concentrations in comparison with the CSR (Contaminated Sites Regulation) standard levels. In the present study, the original soil (B0) was considered as the background soil, since it was collected about 1 km from the main highway intersection and had the same physico-chemical characteristics as the main highway soil. There was no soil that contained no metals in the present study. Hence, the results are compared among plants grown in soils with different metal contamination levels. The outdoor pot experiments were conducted during May – September, 2006 in the Totem Field of the University of British Columbia, in a Completely Randomized Design with five plant species, three different concentrations of multi-metals and three replicates. The five plant species tested were Lolium perenne L (perennial rye grass), Festuca rubra L (red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape). Forty-five plastic pots of diameter 150 mm and height 200 mm were used in each experiment. Two sets of 45 pots were set up for destructive sampling at two stages of plant growth. The weight of each pot without soil was determined before filling it with 1kg soil. The carrier salts for soil spiking with metals were CuSO4, Pb(C2H3O2)2, MnSO4 and ZnSO4. The soil in each pot was mixed with the required concentration of multi-metals in 400 mL of distilled water bringing up the moisture content to the field moisture capacity (36%). This was kept as such for two days for equilibration and seeds sown (0.5 g/pot). The pots were watered for the first two weeks with a complete nutrient solution of N, P, K, Ca and Mg (Hoagland and Arnon, 1950). When the plants were established (after 4 weeks), the pots were transferred to the outdoors to simulate field conditions and watered as needed. The summary of weather conditions during this p Sampling was carried out at two stages of plant growth, 90 and 120 DAS (days after eriod (May 2006 to December 2006) is given in Appendix C, Table 2. sowing). Among the different plant 66  species, Brassica flowered first, so the samples were taken at its maximum flowering stage, 90 DAS and senescence stage, 120 DAS. Even though these stages were different for other species, for consistency and comparability, all species were sampled at the same time to assess their efficiencies for metal uptake. Hereafter, stages of sampling appear in the text, tables and figures as 90 DAS and 120 DAS.  4.2.2 Bio-metric observations Several bio-metric characters such as root length, number of branches per root, shoot length, number of leaves per plant, shoot weight, root weight and root/shoot ratio were recorded at 90 and 120 DAS. Root length and shoot length were measured using a measuring tape.  4.2.3 Laboratory analysis The plant samples were thoroughly washed with running tap water and rinsed with de-ionized water to remove any soil/sediment particles attached to the plant surfaces (Spirochova et al., 2003). Shoots and roots were then separated and oven dried (70ºC) to constant weight. The dried tissues were weighed and ground for analysis of Cu, Pb, Mn and Zn. After dry ashing of plant samples (Lintern et al., 1997), the ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with de-ionized water. Plant extracts were analysed for Cu, Pb, Mn and Zn using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Quality checks and control were performed using blanks, duplicate samples and reference materials.  4.2.4 Statistical analyses The statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. In order to assess the efficiency of plants for phytoextraction and phytostabilisation, the Enrichment Coefficient (EC) of root (Croots/Csoil = ratio of root concentration to soil concentration) and shoot (Cshoots/Csoil = ratio of shoot concentration to soil concentration) and Translocation Factor (TF = Cshoots/Croots = ratio of shoot concentration to root concentration) were calculated (Kumar et al., 1995 and Mattina et al., 2003). Correlation and regression analyses were conducted to establish the 67  relationship between different parameters. The strength of each relationship was interpreted according to the correlation classification of Hopkins (2000), namely negligible: 0.0–0.09; low: 0.1–0.29; moderate: 0.3–0.49; high: 0.5–0.69; very high: 0.7–0.89; nearly perfect: 0.9–1.0. When R was statistically significant at P ≤ 0.05, an asterisk (*) is provided to denote the statistical significance.  4.3 Results and discussion The key characteristics of the studied soil are given in Table 4.2. Table 4.2 Key characteristics of the original soil sample Parameters  Values  Ratio of soil to gravel  1.9  pH in water  5.3  pH in 0.01M CaCl2  4.9  pH in 1M KCl  4.8  Electrical Conductivity  1.7 dS m-1  Total Carbon  1.4 %  Total Nitrogen  0.15 %  Cation Exchange Capacity 21 molc kg-1 Texture  Sandy clay loam  Soil classification  Luvisolic humoferric podsol  The percentage germination of seeds in various treatments after 14 days of sowing is given in Figure 4.1. Poa had the highest germination, followed by Lolium, Brassica, Festuca, and Helianthus. There was no germination in BC soils, whereas the germination in B0 and BA soils was almost the same for all plant species. However, seedlings of Brassica (BrBA) and Helianthus (HBA) did not establish in BA soils, since the plants died one month after germination (i.e. ~45 days after sowing).  68  % germination  120 100  B0  80  BA BC  60 40 20 0 Lolium  Festuca  Helianthus  Poa  Brassica  Plant species  Figure 4.1 replicates.  Germination per cent (mean values). Error bars represent means ±S.D. for three  The difference may be due to the variation among the seeds of the plant species in tolerating different metal concentrations. Similar observations were reported by Adriano (2001) and Wong and Bradshaw (1982), where metal concentrations, even at low levels, can delay or prevent seed germination and establishment. The results discussed in this paper are for B0, BA, LB0 (Lolium B0 soil), LBA (Lolium BA soil), FB0 (Festuca B0 soil), FBA (Festuca BA soil), PB0 (Poa B0 soil), PBA (Poa BA soil), HB0 (Helianthus B0 soil) and BrB0 (Brassica B0 soil). Hereafter these abbreviations appear in the text, figures and tables.  4.3.1 Metal concentrations in plants Two aspects are important in analyzing and interpreting metal accumulations in plants: (1) metal concentration, which is the amount of metal accumulating in plants per unit weight (i.e. mg/kg dry weight) (2) metal content or total metal uptake by plants in a pot, which indicates the metal removal /pot. The metal concentrations in plants (mg/kg) at 120 DAS are given in Figure 4.2 (a) – (d). The plants grown in BA soil had metal concentrations, nearly twice for Cu and Pb, three times for Mn and four times for Zn compared to those grown in B0 soil. None of the plant species studied accumulated metals sufficiently to satisfy the criterion of a hyper-accumulator, as defined by Baker and Brooks (1989), i.e. a metal accumulation exceeding a threshold value of shoot metal concentration of 1% (Zn and Mn) and 0.1% (Cu and Pb) of the dry weight shoot biomass.  69  Pb concentration (mg/kg)  Cu concentration (mg/kg)  120 100  (a)  80 60 40 20 0 1  2  3  4  5  6  7  8  120 100  (b)  80 60 40 20 0 1  2  3  1200 1000  (c)  800 600 400 200 0 1  2  3  4  5  5  6  7  8  6  7  8  Treatments  Zn concentration (mg/kg)  Mn concentration (mg/kg)  Treatments  4  6  7  600 500  (d )  400 300 200 100 0  8  1  Treatments  2  3  4  5  Treatments  Root  Shoot  Figure 4.2. Metal concentrations in plants at 120 DAS. (a) Cu, (b) Pb, (c) Mn, (d) Zn. (1. LB0, 2. LBA, 3. FB0, 4. FBA, 5. HB0, 6. PB0, 7. PBA, 8. BrB0). Error bars represent means ±S.D. for three replicates. Plants with the highest metal concentrations, were Festuca for Cu (FB0 and FBA), Helianthus for Pb and Zn (HB0) and Poa for Mn (PB0 and PBA) as shown in Figure 4.2 (a), (b), (c) and (d) respectively. Poa (PBA) for Mn, differed significantly (P ≤ 0.05) from the other plant species in BA, whereas Festuca (FB0) for Cu had significant differences (P ≤ 0.05) compared to other plant species in B0 (Figure 4.2). Cu, Pb, Mn and Zn concentrations in plants at 120 DAS were higher than those at 90 DAS, and the root concentrations were higher than the shoot concentrations except in Poa for Mn and Helianthus and Brassica for Zn. There was an increase of 5 - 20% Cu, 2 - 10% Pb, 5 – 20% Mn and 11 – 15% Zn at 120 DAS compared to 90 DAS. Similar observations of high Cu accumulation in Helianthus roots were made by Lin et al. (2003) who found that nearly 60% of the total Cu in the roots of Helianthus annuus L. was bound to the cell-wall fraction and the plasma membrane. 70  Cu concentrations reported by Stoltz and Greger (2002) and Shu et al. (2002) in wetland plant species and Paspalum distichum at maturity are higher than the values measured in the present study, possibly due to variations of the plant species and levels of Cu contamination of soils.  4.3.2 Metal content in plants The metal content or the uptake by the plants (root, shoot and total) in a pot appears in Table 4.3. The metal uptake, which denotes the total metal extraction or removal by plant, is more important for assessing efficiencies of plants for phytoremediation than metal concentration in plant (mg/kg).  Lolium recorded a significantly higher uptake (P ≤ 0.05) for all metals at 120 DAS than the other plant species studied, which may be attributed to the high biomass (both root and shoot) exhibited by this species (Table 4.3). The metal uptake at 120 DAS was found to be significantly higher (P ≤ 0.05) than that at 90 DAS for both root and shoot. The metal uptake or removal by plants was found to increase with increasing soil metal loading (Table 4.3). This is mainly due to the high metal concentration in the tissues (Figure 4.2), since the biomass yields were similar for the two loadings (i.e. for B0 and BA soils).  Pb uptake by Helianthus and Brassica grown in B0 soil was higher than that by Lolium, Poa and Festuca grown in both B0 and BA soils at 90 DAS. This may be due to the differences among the plant species in Pb uptake. Also a high total Pb concentration in the soil does not necessarily result in high Pb concentrations in the plants due to the insoluble and immobile nature of Pb in soil (Blaylock et al., 1997). Helianthus has been reported to concentrate Pb in the leaf and stem indicating that it has the prerequisites of a hyper-accumulator plant (Boonyapookana et al., 2005). However in the present study, the concentration of Pb in Helianthus roots was higher than in shoots, both at 90 and 120 DAS, possibly due to the interactive effects of other metals. Even though the metal concentrations of plants in the present study were in the phyto-toxic range according to Levy et al. (1999), no marked visual changes in plant growth were observed.  71  Table 4.3. Metal uptake (µg/pot) by plants (roots, shoots and total) at 90 and 120 DAS Treatments  90 DAS Shoot 37.84 82.10 23.07 31.11 68.78 32.07 36.88 39.39  Total 54.00b 104.53d 39.99a 55.11b 77.80c 42.08a 57.88b 47.61a  Root 74.10 97.50 48.60 47.60 15.60 33.70 52.29 11.55  120 DAS Shoot 89.60 102.90 34.80 44.90 85.00 65.00 83.30 52.50  Total 163.70d 200.40e 83.40b 92.40b 100.60b 98.70b 135.59c 64.05a  Cu  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  Root 16.16 22.44 16.92 24.00 9.03 10.02 20.99 8.22  Pb  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  20.69 23.00 18.51 22.97 16.36 14.04 22.96 16.89  20.3 52.29 11.22 11.93 63.00 17.22 20.97 37.81  40.99ab 77.99c 29.73a 34.90a 79.36c 31.26a 53.14b 66.10c  97.47 92.43 49.32 45.63 23.32 45.44 60.42 19.46  50.68 48.51 16.62 14.90 57.75 37.25 41.82 43.50  148.15d 140.94d 65.94a 60.53a 81.07b 82.69b 102.24c 62.96a  Mn  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  91.68 292.32 75.96 230.72 62.35 96.90 286.20 81.94  236.60 1207.50 88.04 281.01 510.00 261.60 746.12 402.80  328.28a 1499.82b 164.00a 511.73a 572.35a 358.50a 1032.32b 484.74a  290.07 988.00 204.30 427.00 87.75 296.40 700.52 86.10  635.00 1453.20 147.90 387.09 527.50 618.80 1482.40 562.50  925.07b 2441.20c 352.20a 814.09a 615.25a 915.20b 2182.92c 648.60a  164.16a 1211.70e 116.42a 355.08c 401.50c 163.01a 650.88d 240.80b  197.60 683.80 109.80 288.53 45.63 135.63 430.77 346.50  472.00b 1616.20d 213.33a 587.92b 448.13b 425.63b 1248.4d 629.00c Unit - µg/pot n = 3, F values significant at P < 0.05 for all metals. Means followed by a common letter in the same column for each metal do not differ significantly from each other according to the LSD test (P ≤ 0.05). Zn  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  46.56 176.40 41.40 144.96 29.00 44.20 220.32 29.92  117.60 1035.30 75.02 210.12 372.50 118.81 430.56 210.90  274.40 932.40 103.53 297.39 402.50 290.00 817.70 282.50  72  Correlations between metal concentrations in plants at 120 DAS (Table 4.4) revealed a positive interaction or a synergism between the studied metals. In the case of multi-metal contamination, soil analysis results can provide a measure of the ‘availability’ of each metal; but interactions between metals may occur, at the root surface affecting uptake, and within the plant affecting translocation and toxicity (Kabata-Pendias and Pendias, 2001). From the strengths of correlations obtained, it could be seen that maximum interaction was between Cu and Zn (R = 0.899), followed by Zn and Mn (R = 0.706), Pb and Zn (R = 0.618) and Pb and Cu (R = 0.557). According to the correlation classification of Hopkins (2000), correlations were very high for Cu & Zn and Zn & Mn, high for Cu & Pb and Zn & Pb and moderate for Mn & Pb and Cu & Mn (Table 4.4). Table 4.4. Correlations between metal concentrations (mg/kg) in plants (shoot) at 120 DAS Metals  R  Cu & Pb  0.557*  Zn & Pb  0.618*  Mn & Pb  0.467  Cu & Mn  0.458  Cu & Zn  0.899*  Zn & Mn  0.706*  *Correlation coefficient was statistically significant at P ≤ 0.05. The synergism between studied metals reveals that they are unlikely to compete with each other at the site of adsorption, absorption or translocation. This is likely due to low to moderate metal contamination levels in the present study. Further study on metal competition in plant uptake is necessary to confirm the findings in Table 4.4.  Thus based on metal concentration values expressed in mg/kg dry weight, Festuca had the highest accumulation for Cu, Helianthus for Pb and Zn and Poa for Mn. On the other hand, 73  based on uptake values (metal removal/pot), Lolium can be considered as the best candidate among the five studied plants for phytoextraction and Festuca for phytostabilisation. However, metal concentration ratios in plants and soil are the key characteristics that decide the suitability of plant species for phytoextraction/phytostabilisation.  4.3.3 Metal accumulation characteristics The Enrichment Coefficient (EC) and Translocation Factor (TF) values (Kumar et al., 1995 and Mattina et al., 2003) help to identify the suitability of plants for phytoextraction and phytostabilisation. They explain the accumulation characteristics and translocation properties of metals in plants. Significantly higher values of ECshoot (P ≤ 0.05) at 120 DAS were found in Festuca (FB0) for Cu (1.98), Helianthus (HB0) for Pb (0.27) and Zn (5.90), and in Poa (PBA) for Mn (4.15) (Table 4.5). The metal retention in roots, as revealed by ECroot at 120 DAS, was highest in Festuca (FB0) for Cu (2.79), Helianthus (HB0) for Pb (0.72), Poa (PBA) for Mn (4.01) and Lolium (LBA) for Zn (5.1) (Table 4.5). The ability of Poa to accumulate Mn was reported by Liu et al. (2006) in a previous study. Spirochova et al. (2003) reported more Pb retention in the roots of corn plants in a similar study. Pb retention in the root is based on binding to ion exchangeable sites on the cell wall and extracellular precipitation, mainly in the form of lead carbonates deposited in the cell wall (Dushenkov et al., 1995). According to Kumar et al. (1995), a plant suitable for phytostabilisation should have higher EC for roots than for shoots and TF<1. The lowest TF values in the present study were recorded by Lolium for Cu and Pb (LBA at 90 and 120 DAS), Festuca for Mn (FB0 and FBA at 90 and 120 DAS) and Poa for Zn (PB0 and PBA at 90 and 120 DAS). Based on the EC and TF values, for phytoextraction, Festuca was found to be the best for Cu, Helianthus for Pb and Zn and Poa for Mn; whereas for phytostabilisation, Lolium was found to be the best for Cu and Pb, Festuca for Mn, and Poa for Zn.  74  Table 4.5. Translocation Factor (TF) and Enrichment Coefficient (EC) of metals. Treatments  Enrichment Coefficient 90 DAS  120 DAS  Translocation factor 90 DAS  120 DAS  Root Shoot Root Shoot Cu LB0 1.40 1.12 2.08 1.71 0.79 0.82 LBA 1.10 0.66 1.81 1.18 0.69 0.65 FB0 1.88 1.48 2.79 1.98 0.80 0.83 FBA 1.96 1.40 1.90 1.60 0.81 0.73 HB0 1.48 1.30 1.99 1.65 0.88 0.84 PB0 1.28 1.27 1.66 1.61 0.99 0.97 PBA 1.06 0.78 1.78 1.07 0.73 0.77 BrB0 1.12 0.94 1.12 1.04 0.85 0.66 F ns * * ns * * Pb LB0 0.50 0.17 0.53 0.23 0.33 0.35 LBA 0.51 0.16 0.55 0.18 0.31 0.30 FB0 0.57 0.20 0.63 0.22 0.35 0.34 FBA 0.46 0.19 0.54 0.20 0.41 0.37 HB0 0.64 0.29 0.72 0.27 0.45 0.40 PB0 0.46 0.17 0.54 0.17 0.38 0.32 PBA 0.47 0.15 0.55 0.18 0.35 0.32 BrB0 0.55 0.22 0.67 0.21 0.41 0.33 * ns F ns ns * ns Mn LB0 1.40 1.24 1.20 1.70 0.88 1.40 LBA 2.30 1.87 3.40 3.10 0.82 0.91 FB0 1.50 1.02 1.75 1.31 0.67 0.75 FBA 2.69 2.05 3.30 2.64 0.76 0.80 HB0 1.50 1.45 1.74 1.63 0.94 0.93 PB0 2.14 1.80 2.40 2.11 0.84 0.86 PBA 3.12 3.20 4.01 4.15 1.02 1.03 BrB0 1.69 1.49 1.80 1.64 0.87 0.91 F * * * * * * Zn LB0 3.90 3.33 4.22 3.50 0.86 0.84 LBA 3.31 3.24 5.13 4.82 0.99 0.94 FB0 3.72 3.92 4.51 4.31 1.05 0.97 FBA 3.60 3.31 4.20 4.33 0.90 0.91 HB0 3.21 4.82 4.32 5.92 1.49 1.38 PB0 4.13 3.11 4.81 3.81 0.77 0.80 PBA 4.80 3.70 5.02 3.92 0.76 0.78 BrB0 2.91 3.73 3.61 4.02 1.26 1.14 F * * * * * * F values compare the EC and TF values for different treatments (LB0, LBA, FB0, FBA, HB0, PB0, PBA, BrB0) at each stage, 90 DAS and 120 DAS. Mean values. n = 3. ns – F not significant, * - F significant at P ≤ 0.05. 75  Metal-tolerant species with high ECroot and low TF can be used for phytostabilisation of metalcontaminated sites (Yoon et al., 2006). The efficiencies of plants for phytostabilisation are determined by metal retention in roots since there is less translocation of metals to the aboveground portions. This enables harvestable biomass to continue growing uninhibited by metals and further reduces the passage of metals into the food chain through the activity of herbivores (McIntyre, 2003). In plants with TF < 1, there is less translocation of metals to the above-ground portions. The lower translocation of metals to the above-ground portions may be due to immobilization of metals in roots by vacuole sequestration or cell wall binding, thereby preventing interaction with high-molecular-weight compounds in the plant cell cytoplasm (Salt et al., 1995; Macfie and Welbourn, 2000). In the present study, TF was < 1 for all plant species except in Poa (PBA) for Mn and Helianthus (HB0) and Brassica (BrB0) for Zn. It could be seen that for Lolium, when the soil Zn concentration was increased, more Zn was translocated from roots and accumulated in the tops of the plant (TF, 0.99 and 0.94 in LBA, compared to 0.86 and 0.84 in LB0 at 90 and 120 DAS). Zn accumulated in the shoot portion has been reported to be concentrated in chloroplasts, vacuole fluids and cell membranes (KabataPendias and Pendias, 2001). The accumulation of Zn by the above-ground portions is representative of the Zn phytoextraction potential of plants (Hamlin and Parker 2006). Because of the high CEC of plant cell walls, metals are usually transported as chelated species rather than in ionic form. Hence the capacity of Zn to bind with organic anions is an important aspect that controls the plant transport of Zn. Thus based on the results of the present study, Lolium, Poa and Festuca can be recommended for phytostabilisation of soils contaminated with moderate levels of Cu, Pb, Mn and Zn.  4.3.4 Relationships of metal concentration and bio-metric characters of plants The biometric characteristics of plants and plant biomass are important parameters affecting plant health and successful functioning for metal remediation. Based on the correlation and regression analyses of each biometric character (i.e. number of leaves/plant, number of root branches, root length and shoot length) with plant metal concentrations, it is found that root  76  length, shoot length and number of branches/root are the main characteristics that influence the metal concentrations in plants. Table 4.6. Correlations between metal concentrations (mg/kg) in plants and biometric characters at 120 DAS Metal Cu  Root RE R Shoot RE R  Pb  Root RE R Shoot RE R  Mn  Root RE R Shoot RE R  Zn  Root RE R Shoot RE R  Root length  Shoot length  No. of branches/root  y = -4.4208x + 121.01  y = -0.8298x + 101.73  y = 0.5252x + 51.088  0.565*  -0.255  0.564*  y = -3.618x + 78.014  y = -0.6625x + 61.682  y = 0.9447x + 10.998  0.509  -0.629*  0.518  y = -2.8878x + 91.446  y = -0.2825x + 70.192  y = 0.7057x + 3.8408  0.487  0.620*  0.708*  = -0.6775x + 27.436  y = -0.0493x + 21.882  y = 0.9296x + 34.144  0.227  0.156  0.323  y = -72.053x + 1251.7  y = -13.133x + 1006.1  y = -6.9427x + 435.41  -0.599*  0.647*  -0.477  y = -64.302x + 1093.7  y = -11.231x + 801.58  = -2.2643x + 265.9  -0.022  0.565*  -0.286  y = -50.455x + 838.21  y = -7.8505x + 564.26  y = -0.7197x + 170.77  -0.131  0.701*  0.189  y = -45.199x + 757.61  y = -6.7467x + 509.92  y = 4.1943x + 66.159  -0.503  0.643*  0.3469  R - Correlation coefficient, RE - Regression Equation. * R was statistically significant at P ≤ 0.05. 77  Table 4.6 summarizes the relationship between metal concentrations and plant biometric characters at 120 DAS. Root parameters such as root length and number of branches/root have a significant influence (P ≤ 0.05) on root and shoot concentrations of Cu and Pb, whereas, Mn and Zn concentrations are significantly influenced by shoot length. In the case of Pb, a significant and positive correlation is obtained between root concentration and number of branches/root (R = 0.708). Root parameters control the efficiency of rhizosphere metal dynamics and ultimately the efficiency of plants for soil metal de-contamination (Uren and Reisenauer, 1998; Marschner et al., 1995). The amount and composition of root exudates released into the rhizosphere are highly variable and dependent on plant species, stage of plant growth, physico-chemical environment and metal toxicity stress (Marschner et al., 1995). This accounts for the differential absorption of metals by plant species at different growth stages and different soil contamination levels in the present study.  4.3.5 Relationships of metal content and biomass of plants The metal contents of plants per pot at 120 DAS are plotted against biomass levels in Figure 4.3. Significant and positive correlations (P ≤ 0.05) are obtained between the Pb content of roots and root biomass (R = 0.909), Pb content of shoots and shoot biomass (R = 0.902), Cu content of roots and root biomass (R = 0.814), Cu content of shoots and shoot biomass (R = 0.705) (Figure 4.3). Mn and Zn contents in plants have only nonsignificant correlations with the plant biomass for both root and shoot, and the values are not presented in Figure 4.3. The very low metal content in plants in the present study compared to many previous studies may be due to low biomass accumulation per pot. This could be due to the fact that, in this study, seeds were directly sown in the multi-metals contaminated soil and their survival ability and metal uptake pattern could have been affected as discussed above. Most previous studies on phytoremediation were conducted in hydroponic cultures (e.g. Tandy et al., 2006; January et al., 2008), or seeds were sown in potting mix or sand mix and the seedlings transplanted to the contaminated soils (e.g. Marchiol et al., 2004; Bhattacharya et al., 2006). Low levels of metal accumulation may also be due to interactions between metal ions in multi-metal-contaminated soils, inhibiting  78  metal uptake (Ebbs and Kochian, 1997), relative to soils having elevated levels of single metals  120 100 80 60 40 y = 28.674Ln(x) + 61.447 R = 0.814  20 0 0  0.5  1  1.5  2  Cu content in shoot (micro grams/pot)  Cu content in root (micro grams/pot)  (Huang and Cunningham, 1996; Blaylock et al., 1997).  120 100 80 60 40 y = 25.444Ln(x) + 59.131 R = 0.705  20 0  2.5  0  120 100 80 60 40 y = 31.954Ln(x) + 69.598 R = 0.909  20 0 0  0.5  1  1.5  2  2  3  Shoot biomass (g/pot)  Pb content in shoot (micro grams/pot)  Pb content in root (micro grams/pot)  Root biomass (g/pot)  1  2.5  Root biomass (g/pot)  70 60 50 40 30 20  y = 20.989Ln(x) + 30.119 R = 0.902  10 0 0  1  2  3  Shoot biomass (g/pot)  Figure 4.3. Relationship between metal content in plants and plant biomass (dry weight) at 120 DAS. (a) Root biomass and Cu content in roots. (b) Shoot biomass and Cu content in shoots. (c) Root biomass and Pb content in roots. (d) Shoot biomass and Pb content in shoots. Such interactions could also explain why Brassica napus and Helianthus annuus accumulated less Pb and Zn in shoots than those reported by previous authors (Huang and Cunningham, 1996; Ebbs et al., 1997). In this study, Lolium recorded the highest values for shoot and root biomass (2.8 and 1.9 g/pot) at 120 DAS. However, Festuca recorded the highest root/shoot ratio, followed by Poa. Root biomass and root/shoot biomass ratio are important aspects of phytostabilisation, since the efficiency of rhizosphere metal precipitation depends on the exposure of plant roots to the contaminated zones. Lolium, Poa and Festuca have been identified as being suitable for phytostabilisation of metalcontaminants (Cu, Pb, Mn and Zn) in highway soils. Growing these plant species along highway 79  soils could also help to reduce the metal concentration of highway runoff by filtering or trapping metal-containing particulates and reducing the amount of metal-contaminated sediments entering the biota. It is suggested that these plants be tested under field conditions.  4.4 Conclusions and recommendations  •  The germination of seeds is affected by the metal concentrations. In BC soils (total Cu, Pb, Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg, respectively), none of the seeds germinated because of metal toxicities.  •  Plant metal concentrations depend on the original soil metal concentrations. The metal concentrations in plants increased by nearly a factor of 2 for Cu and Pb, three times for Mn and four times for Zn in BA soils, compared to B0 soils.  •  The efficiency of plants to accumulate metals followed the order, Festuca > Lolium > Helianthus > Poa > Brassica for Cu, Helianthus > Brassica > Festuca > Poa > Lolium for Pb, Poa > Festuca > Lolium > Brassica > Helianthus for Mn and Helianthus > Festuca > Poa > Brassica > Lolium for Zn. There was an increase of 5 - 20% Cu, 2 - 10% Pb, 5 – 20% Mn and 11 – 15% Zn in plants at 120 DAS compared to 90 DAS.  •  The plant biomass plays a significant role in total metal removal by plants. Lolium recorded a significantly higher uptake (P < 0.05) for all the metals than the other plant species studied, as Lolium had the highest biomass (both root and shoot), whereas Festuca recorded the lowest uptake for all four metals.  •  Metal retention in roots, as revealed by the values of ECroot at 120 DAS, was highest in Festuca (FB0) for Cu, Helianthus (HB0) for Pb, Poa (PBA) for Mn and Lolium (LBA) for Zn. Based on the EC (Enrichment Coefficient) and TF (Translocation Factor) values, for phytoextraction, Festuca was found to be the best for Cu, Helianthus for Pb and Zn and Poa for Mn. For phytostabilisation, Lolium was found to be best for Cu and Pb, Festuca for Mn, and Poa for Zn.  •  The effects of combined metals on plant metal uptake are complex, and further study is required.  80  •  Lolium, Poa and Festuca are suggested for pilot study of highway roadside soil remediation to evaluate if they can mitigate the metal toxicity of highway runoff by sequestering metal contaminants in roots or rhizosphere. In the field, interactions of various pedogenic and biogenic factors need to be considered.  •  Improvements to phytostabilisation could be obtained by species mixtures and implementing soil management practices that have a positive influence on the efficiency of this process. The presence and concentration of the appropriate transport proteins or translocating chelating molecules in plants play a very important role in the translocation of metals. These factors should be investigated in future studies.  81  4.5 References Adriano, D. C. (2001). 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(1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612. Spirochova, I. K., Pucocharova, J., Kafka, Z., Kubal, M., Soudek, P. and Vanek, T. (2003). Accumulation of heavy metals by in vitro cultures of plants. Water Air Soil Pollut., 3, 269276. Stoltz, E. and Greger, M. (2002). Accumulation properties of As, Cd, Cu, Pb and Zn by four wetland plant species growing on submerged mine tailings. Environ Exp Bot., 47, 271– 280. Tandy, S., Schulin, R. and Nowack, B. (2006). The influence of EDDS on the uptake of heavy metals in hydroponically grown sunflowers. Chemosphere, 62(9), 1454-1463. Uren, N. C. and Reisenauer, H. M. (1998). The role of root exudates in nutrient acquisition. Adv. Plant Nutr., 3, 79–114. Weng, G., Wu, L., Wang, Z., Luo, Y. and Christie, P. (2005). Copper uptake by four Elsholtzia ecotypes supplied with varying levels of copper in solution culture. Environment International, 31 (6), 880-884. Wong, M. H. and Bradshaw, A .D. (1982). Comparison of the toxicity of heavy metals, using root elongation of rye grass, Lolium perenne. New Phytol., 91, 255-261. Yoon, J., Cao, X., Zhou, Q. and Ma, L. Q. (2006). Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site. The Sci. Total Environ., 368, 456 –464.  85  5.  4  EXPLORATION OF PHYTOREMEDIATION AND ITS EFFECT ON MOBILITY OF METALS IN SOIL – A FRACTIONATION STUDY  5.1 Introduction Pollution of the natural environment due to the release of metals from various sources is a global problem (Kabata-Pendias and Pendias, 2001). Sources of anthropogenic metal contamination include smelting of metalliferous ore, electroplating, gas exhaust, energy and fuel production, application of fertilizers and municipal sludge to land, industrial manufacturing and vehicular traffic (Singh et al., 2004; Sansalone et al., 1996). In urban areas, vehicular traffic is an important source of metal contamination in the environment (REC, 1998). High concentrations of metals, particularly lead, zinc, manganese, and copper in highway runoff result from the wear of brakes, tires and other vehicle parts, leakage of lubricants (Birch and Scollen, 2003; Sutherland et al., 2003) and exhaust emissions (Preciado and Li, 2006). Even though Mn is ubiquitous as a hydrous oxide, it was studied because of high Mn concentrations in roadside soils which can interfere with the normal life processes. Mn is from the methyl cyclopentadienyl manganese tricarbonyl (MMT), replacement of the tetraethyl lead (TEL) used as an anti-knock compound for gasoline engines in the early 1980s. Li et al. (2008) indicates that the concentration of Mn increased in the stream sediments and highway runoff. Metals are the most persistent constituents found in pavement runoff, and the transport of anthropogenic metal constituents by pavement runoff can adversely affect the quality of adjacent receiving waters and soils (Sansalone et al., 1996; Fakayode and Olu-Owolabi, 2003, Li et al., 2008). Exposure to high levels of these metals has been linked to adverse health effects on humans and wildlife (Bozkurt et al., 2000). The environmental hazards of metal pollution in soil depend on the geochemical and biochemical properties of a given metal and are related to pedogenic and biogenic processes operating over time, which determine their mobility and bioavailability (Adriano et al., 2004; Kabata-Pendias and Pendias, 2001). Water-soluble and exchangeable forms of metals in soils are 4  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2009) Exploration of phytoremediation and its effect on mobility of metals in soil – a fractionation study. Land Contamination and Reclamation, 17(2): 223-236.  86  considered readily mobile and available to plants, whereas metals incorporated into crystalline lattices of clays appear to be relatively inactive (Shuman, 1985). Other forms of metals, precipitated as carbonate, occluded in Fe, Mn, and Al oxides, or complexed with organic matter, could be strongly bound in soils, depending on the actual composition, physical and chemical properties of soil (Li and Thornton, 2001). The remediation of metal-contamination in soils along highways extending to thousands of kilometers is challenging. Existing metal remediation techniques such as solidification/stabilization, flotation, soil washing, electroremediation, bioleaching and microbial surfactants (Singh and Cameotra, 2004; Zoubeir et al., 2007) are expensive and not feasible for extended distances and large contaminated areas. Phytoremediation is a relatively sustainable and eco-friendly method not only to reduce the hazard associated with the presence of excess metals, but also to improve the soil quality and restore its functionality (Salt et al., 1995; Ebbs and Kochian, 1998; Singh et al., 2004). Using phytostabilisation to reduce the environmental impact by holding the metal-pollutants at the source location in non-mobile forms by the growth of plants (Smith and Bradshaw, 1979) could be a feasible management method for soils along highways, because it requires minimal maintenance. Most phytoremediation studies have focused only on the plant-uptake of metals at only one stage of plant growth. Even though it is known that plant roots exude organic compounds which mobilize metals in soils and have indirect effects on microbial activity, in turn, affect the soil properties (Uren and Reisenauer, 1988), there has been limited study on the effects of phytoremediation on metal fractions in soil. Only a few studies have focused on the redistribution of metals in various soil fractions due to plant growth, for example, Guptha and Sinha (2006) on Sesamum indicum, King et al. (2008) on the mobilisation of Zn and Pb by the growth of Salix, Populus and Alnus and Degryse et al. (2008) on the mobilisation of Zn and Cu by growing spinach (Spinacia oleracea L.) and tomato (Lycopersicon esculentum L.). There is no published study to the authors’ knowledge which examines the mobility or immobility of metals in soils at different stages of plant growth during phytoremediation. The quantity of metal associated with a particular soil fraction varies depending upon plant species, stage of plant growth, conditions of the site and plant uptake characteristics (King et al., 2008). These factors control the potential mobility of metals to water sources and food chain, causing health and environmental hazards (Adriano et al., 2004; King, 2008). Therefore, the present study is 87  focused on assessing the effect of growth of five different plant species on the redistribution of soil metal fractions for Cu, Pb, Mn and Zn at two growth stages in roadside soil. The study involved a systematic and comprehensive effort with the research protocol: (1) investigating the physico-chemical properties of the soil before and after plant growth, (2) assessing the distribution of metal fractions in the rhizosphere at two different growth stages under variable multi- metal contamination levels, (3) examining the relationship of soil-metal fractionation to physico-chemical properties of soil (4) assessing the plant-metal accumulation at different growth stages; and (5) examining the relationship of plant metal concentrations with soil-metal fractionation. This study provides information on the effect of plant growth on metal dynamics and the distribution of metal fractions in soil at two stages of plant growth. The results identify suitable plants for highway soil metals remediation.  5.2 Materials and methods  The studied soil was collected from the grassed and vacant yard of Surrey Fire Hall No. 5, located 1 km north of the intersection of TCH (Trans Canada Highway) with the 176 Street overpass in Surrey, British Columbia. This site has the same soil as the nearby highway intersection (Luttmerding, 1980). Soils with two different metal concentrations were studied: (a) B0, the original soil containing concentrations of Cu 52 mg/kg, Pb 93 mg/kg, Mn 215 mg/kg, Zn 70 mg/kg. (b) BA, the original soil spiked with addition of all four metals to give total Cu, Pb, Mn, and Zn concentrations of 80, 146, 408 and 148 mg/kg, respectively. The metal concentrations studied were based on the results from the preliminary studies of the highway site (Padmavathiamma et al., 2007), previous work on roadside soils (Fakayode and Olu-Owolabi, 2003; Preciado and Li, 2006) and the British Columbia Standards for B.C contaminated sites (B.C Ministry of Environment, 1995). Metal fractionation studies were conducted in soils before and after plant growth in pot experiments. The five plant species tested are common in temperate maritime climate, typically found along the west coasts at the middle latitudes of the world's continents and are also listed in the data base "Phytorem" (Environment Canada, 2003). They are Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape). They were grown in B0 and BA soils separately. The detailed experimental program of the study is summarized in Table 5.1. Details of soil spiking, experiment layout and sampling are given in 88  chapter 4, section 4.2.1. At each sampling stage, i.e. at 90 and 120 DAS, destructive sampling was applied. After removing the plants, both shoot and root, the soil in the pot was taken out, mixed well and a representative sample was taken. Since the roots covered almost the full soil, the whole soil was considered as the rhizosphere soil, and there is no bulk soil. The soil samples were taken to the laboratory in an ice bucket and immediately refrigerated at 4°C. Metal fractionation in the soil was performed after estimating the moisture content of soil samples. The plant samples were thoroughly washed with running tap water and rinsed with de-ionized water to remove any soil/sediment particles attached to the plant surfaces. Shoots and roots were then separated and oven dried (at 70ºC) to constant weight. The dried tissues were weighed and ground for analysis of Cu, Pb, Mn and Zn. Table 5.1 Experimental Program for identification of plant species for phytostabilisation Metals studied  Metal concentrations  Plant species  Stages of sampling  Chemical Analysis  Cu  B0 – Original soil with metal concentrations of 52, 93, 215 and 70 mg/kg of Cu, Pb, Mn and Zn respectively.  Lolium perenne L (perennial rye grass)  90 DAS  Basic soil characteristics.  Pb Mn Zn  BA – B0 soil spiked with metals to give total metal concentrations of 80, 146, 408 and 148 mg/kg of Cu, Pb, Mn and Zn respectively.  Festuca rubra L (creeping red fescue)  120 DAS Metal fractionation by Selective Sequential Extraction. Total soil metals.  Helianthus annuus L (sunflower) Poa pratensis L (Kentucky bluegrass)  Plant metal concentrations. Plant uptake  Brassica napus L (rape) Experimental Design – Completely Randomized Design, 10 treatments (5 plant species and 2 metal concentrations) and 3 replications. Soil and plant samples were collected at two stages of plant growth, 90 and 120 DAS days after sowing. Fallowed soils without plants, subjected to similar conditions like other treatments 89  were also kept for comparison and sampled at 90 and 120 DAS. The basic characteristics of the soil such as pH, electrical conductivity, texture, % Carbon, total N and CEC were measured based on standard procedures (Table 3.1). The total metals in the soil samples were estimated by the USEPA method (Smoley, 1992). Different metal fractions in soils: exchangeable, oxide, organic and residual fractions were estimated using selective sequential extraction, according to the procedure of Tessier et al. (1979), as modified by Preciado and Li (2006). Since the soil has a pH <6, the carbonate fraction was negligible. Hence, the carbonate extraction was combined with oxide extraction and reported as such. The plant samples were air dried and ashed as recommended by Lintern et al (1997). The ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with deionized water. Soil and plant extracts were analysed for Cu, Pb, Mn and Zn using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Blanks, duplicate samples and reference materials were used to ensure the quality and accuracy of the chemical analysis of the plants and soils. The statistical significance of differences among means was determined by oneway analysis of variance (ANOVA) to compare the treatment effects on soil metal fractionation, total soil metal concentration as well as metal uptake by plants. Statistical significance was defined at the level of P ≤ 0.05. Correlation and regression analyses were conducted to establish the relationship between soil and plant metal concentrations. The strength of each relationship was interpreted according to the correlation classification of Hopkins (2000), namely negligible: 0.0–0.09; low: 0.1–0.29; moderate: 0.3–0.49; high: 0.5– 0.69; very high: 0.7–0.89; nearly perfect: 0.9–1.0. When R was statistically significant at P ≤ 0.05, an asterisk (*) is provided to denote the statistical significance.  5.3 Results and discussion Basic characteristics of the soils studied are summarized in Chapter 4, Table 4.2. All plant species tested (Lolium, Festuca, Helianthus, Poa and Brassica) survived in B0, whereas only three (Lolium, Poa and Festuca) survived in BA. LB0 represents Lolium in B0 soil, LBA (Lolium in BA soil), FB0 (Festuca in B0 soil), FBA (Festuca in BA soil), PB0 (Poa in B0 soil), PBA (Poa in BA soil), HB0 (Helianthus in B0 soil) and BrB0 (Brassica in B0 soil). Hereafter these abbreviations are used in the text, figures and tables.  90  5.3.1 Physico-chemical properties of soil as influenced by plant growth The pH and electrical conductivity of soils at 90 and 120 DAS are given in Figure 5.1. The initial pH values for B0 and BA soils were 5.3 and 5.0. After fallowing and maintaining the same moisture % as the treated plots, the pH was found to be 5.2 and 5.0 for B0 and 4.9 and 4.8 for BA soil at 90 and 120 DAS. With plant growth, soil pH ranged from 4.9-5.3 at 90 DAS and 5.3-5.7 at 120 DAS. Thus there was a decrease in soil pH at 90 DAS and an increase at 120 DAS from the initial value with plant growth, while in fallowed soils a decrease from the initial value was observed at both 90 and 120 DAS. The decrease in soil pH in fallowed soils and planted soil at 90 DAS may be due to re-wetting of air-dried soil leading to microbial stimulus and enhanced reactions such as nitrification. The increase in soil pH at 120 DAS in planted soils, when compared to fallowed soils, may be attributed to the effect of root exudates or microbial exudates in the rhizosphere, which are important from the phytostabilisation point of view. The decrease in electrical conductivity accompanied by plant growth may be due to the lowering of soluble nutrient and metal ions in the soil by plant absorption or partitioning in insoluble soil fractions. Variations in pH of soils grown with different plant species may be due to the differential uptake of cations and anions by the plants, coupled with release of H+ or HCO3- and OH- (Marschner, 1995). Soil pH is the “master variable” due to its potential to modify metal solubility / availability in many ways (McBride, 1994). It controls dissolution / precipitation reactions, regulates the ionisation of pH-dependent exchange sites on organic matter and oxide clay minerals and influences metal speciation in soils (Adriano et al., 2004; Conesa et al., 2006).  91  6.0 90 DAS  (a)  120 DAS  Soil pH  5.5  5.0  4.5 LB0  FB0  HB0  PB0 BrB0  LBA  FBA PBA  Electrical Conductivity (dS/m)  Treatment 2.0 90 DAS  (b)  120 DAS  1.5  1.0  0.5  0.0 LB0  FB0  HB0  PB0 BrB0  LBA FBA PBA  Treatment Fallowed soil Initial value 90 DAS 120 DAS Figure 5.1 (a) pH and (b) Electrical Conductivity of soils at 90 and 120 DAS. Error bars represent ±S.D of means of three replicates. F-values for pH and Electrical Conductivity are significant at P <0.05. Fallowed soils are soils without plants, but subjected to similar conditions like other treatments and sampled at 90 and 120 DAS. 92  5.3.2 Metal fractionation in Soils The metals in the soils were categorized in four fractions: exchangeable, oxide and carbonatebound, organic-bound and residual. The partitioning of Cu, Pb, Mn and Zn in fallowed soils (without plant growth) and in soils with plant growth at 90 and 120 DAS is presented in Figure 5.2. The exchangeable fraction was found to be higher in BA soil than in B0 soil for all four metals. The % distributions of metal fractions in fallowed soils are as follows: Cu mainly in organic and residual fractions, Pb in oxide and residual fractions, Mn in oxide fraction, Zn in exchangeable and oxide fractions. The changes in metal fractionation with plant growth are discussed below.  Cu – The results show that Cu was retained mainly in the organic fraction at 90 DAS (days after sowing) and oxide fraction at 120 DAS (Figure 5.2). Compared to B0 soil (4% exchangeable Cu), there was a significant reduction (P<0.05) of exchangeable Cu (<1%) at 90 and 120 DAS due to the growth of Festuca (FB0 and FBA). In soils growing Festuca (FB0 at 90 DAS, FB0 and FBA 120 DAS), more than 90 % of exchangeable Cu was re-distributed to oxide Cu (see Figure 5.2), indicating the suitability of Festuca as a phytostabilising plant for Cu. There was an increase of exchangeable Cu in soils growing Poa (7%) and Brassica (6%) at 120 DAS. This is not desirable because of the increased mobility and bioavailability of this fraction. The increase of exchangeable Cu in Poa and Brassica growing soils can be attributed to the effect of root exudates and extruded protons in decreasing the soil pH and increasing the solubility of Cu. The organic Cu in soils growing Lolium (LB0) and Poa (PB0) were 45 and 47% at 90 DAS, whereas it was only 27-32% in soils growing Festuca (FB0), Helianthus (HB0) and Brassica (BrB0). This may be due to the well developed fibrous root system of Lolium and Poa, contributing to high soil organic matter and hence to organic-bound Cu in the soil. This is consistent with the observations of several other authors (Balasoiu et al., 2001 and Clemente et al., 2006), indicating that Cu forms very stable complexes with organic matter. Cu associated with the organic fraction can be mobile or immobile depending on the organic fraction to which it is complexed. The acidic pH of the soil in the present study (4.7 – 5.2 at 90 DAS) tended to retain less Cu as stable organic complexes, re-distributing the organic fraction to the oxide fraction at 120 DAS. This may explain the high partitioning of Cu in the oxide fraction at 120 DAS, regardless of the plant species. The differential partitioning of Cu in various soil fractions during the growth of different plant species can be attributed to the differential release of root exudates. These root 93  secretions have the ability to mobilize metals by shifting the equilibrium between different forms (Kabata-Pendias and Pendias, 2001). The ways in which plant roots alter the local metal chemistry in the rhizosphere have an impact on changing the oxidation state of metals (Marschner, 1995).  Pb- Pb partitioning through sequential extractions showed that a minor fraction of metal was exchangeable and that complexation with oxides, together with organic matter, was high. Although there was an increase in the organic fraction of Pb due to plant growth, the oxide fraction dominated at 90 DAS, and increased further at 120 DAS by re-distribution from exchangeable and organic forms (Figure 5.2). This may be due to the effect of plants in enhancing Pb immobilisation through stimulation of microbial activity, uptake into roots, redox reactions and formation and precipitation of insoluble Pb compounds in the rhizosphere (Wenzel et al., 1999). Poa soils (PB0 and PBA) had the least exchangeable Pb (1%) at both 90 and 120 DAS. The highest exchangeable Pb was observed in soils growing Helianthus (HB0) and Brassica (BrB0) at 120 DAS (Figure 5.2). As in the case of Cu, the highest oxide Pb was observed in Festuca-growing soils (FB0 and FBA) at 120 DAS and organic Pb in soils growing Lolium (LBA) and Poa (PBA) at 90 DAS. The re-distribution of more than 90 % of soluble or mobile (exchangeable) Pb to insoluble or immobile (oxide) Pb by the growth of Poa (PB0 and PBA at 120 DAS) reveals the suitability of Poa as a phytostabilising plant for Pb. Pb is similar in behaviour to Cu with respect to its tight bonding with the relatively insoluble fractions of the soil. However, Pb does not form stable organic complexes and hence the relative distribution in organic fraction was lower than that of Cu (Li et al., 2007). This is consistent with McBride and Martınez (1994) who reported that the main mechanisms responsible for lead immobilisation are chemisorption on oxides and silicate clays and precipitation as carbonates, hydroxides or phosphates.  Mn - The dominance of Mn in the oxide fraction with very little partitioning in the organic fraction occurred at 90 and 120 DAS for each of the plant species. Poa (PB0 and PBA) tended to retain more Mn in the exchangeable and oxide fractions of the soil, compared to the other plant species. As the growth stage advanced from 90 to 120 DAS, the exchangeable Mn increased further in soils growing Poa (PB0 and PBA) and Festuca (FB0 and FBA), whereas it decreased significantly (P<0.05) in Lolium-growing soils (LB0 and LBA, Figure 5.2). The low content of exchangeable Mn in Lolium-growing soils is notable, since the mobility of metal fractions is in 94  the order, exchangeable > carbonate specifically adsorbed > Fe-Mn oxide > organic sulfide > residual (Li and Thornton, 2001). Thus Lolium appears to be a suitable plant for phytostabilising Mn. The oxide fraction of Mn dominated in Festuca-growing soils (FB0 and FBA) at both 90 and 120 DAS. At 120 DAS, the organic fraction of Mn was further reduced by redistribution to the oxide and exchangeable fractions (Figure 5.2). This may due to the fact that Mn in soil is largely associated with fulvic acid, and the Mn2+ bound to these compounds is highly ionized (Cheshire et al., 1977).  Zn - The predominance of Zn in the exchangeable and oxide fraction is evident from Figure 5.2. As the growth stage advanced from 90 to 120 DAS, the proportion of the oxide and organic fractions increased as a result of re-distribution from exchangeable and residual fractions. The exchangeable fraction of Zn was relatively high at 90 DAS, with a decline at 120 DAS (Figure 5.2). In Festuca growing soils (FBA), the exchangeable fraction decreased from 22% at 90 DAS to 14 % at 120 DAS. A significant reduction (P<0.05) in exchangeable Zn was found in soils growing Poa (PB0 and PBA) both at 90 and 120 DAS, giving evidence of the suitability of Poa for phytostabilisation of Zn. Unlike other metals which partitioned predominantly among oxides, organic and residual phases, Zn seems to be more in the exchangeable form (Alvarez et al., 2002; Su and Wong, 2003). The acidic pH, together with secretion of root exudates, might explain the high proportion of Zn associated with the exchangeable fraction. Since the pH was acidic for the soils examined (4.7-5.2 at 90 DAS and 5.3-5.7 at 120 DAS), there is greater risk of Zn mobilization compared to the other metals studied.  95  60% 40% 20%  80% 60% 40% 20% 0%  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  40% 20%  80% 60% 40% 20% 0%  0%  40% 20%  80% 60% 40% 20% 0%  0%  60% 40% 20%  20%  80% 60% 40% 20%  80% 60% 40% 20%  90 DAS  120 DAS Oxide  80% 60% 40% 20% 0%  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  BA  100%  0%  0%  Exch.  40%  B0  Zn fractionation (%)  Zn fractionation (%)  80%  BA  60%  0%  100%  100%  B0  80%  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  BA  100% Mn fractionation (%)  Mn fractionation (%)  60%  B0  0%  100%  80%  20%  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  100%  40%  100% Pb fractionation (%)  Pb fractionation (%)  Pb fractionation (%)  60%  60%  0%  100%  80%  80%  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  100%  Mn fractionation (%)  Cu fractionation (%)  80%  0%  Zn fractionation (%)  100%  100%  Cu fractionation (%)  Cu fractionation (%)  100%  Organic  B0  BA  Fallowed soil  Residual  Figure 5.2 Metal fractionation (%) in soil by the influence of plant growth at 90 and 120 DAS. n = 3, F-values are significant at P <0.05. LB0 (Lolium B0 soil), LBA (Lolium BA soil), FB0 (Festuca B0 soil), FBA (Festuca BA soil), PB0 (Poa B0 soil), PBA (Poa BA soil), HB0 (Helianthus B0 soil) and BrB0 (Brassica B0 soil).  96  5.3.3 Metal comparison The metal fractionation studies summarized above indicate a significant partitioning of metals to insoluble forms by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. There was a decrease in the exchangeable fraction and an increase in the oxide and organic fraction of metals in soils as a result of plant growth. However the increase was more in the organic fraction for Cu and Pb and in the oxide fraction for Mn and Zn. This indicates that Cu and Pb are better able to form stable organic complexes than Mn and Zn for the range of conditions investigated. Thus the dynamic metal fractions were exchangeable, oxide and organic, while the residual fraction was not significantly affected by plant growth. Since the aim of the present study was to explore phytoremediation based on mobility/bioavailability of soil metals at different stages of plant growth, the total metal concentration values of soil obtained after harvesting plants are less significant, and the decrease observed often lies within the range of experimental error (Calace et al., 2002). However, a comparison of final metal concentrations in soils grown with the five plant species shows that maximum reduction was achieved by the growth of Lolium for Cu and Pb, Poa for Mn and Lolium and Brassica for Zn. The ability of Poa pratensis to phytoremediate Mn-contaminated soil was reported previously by Liu et al. (2006), while the effect of Brassica napus in reducing soil Zn was reported by Ebbs et al. (1997) and Ebbs and Kochian (1998). If the total metal concentration in soil is taken as a criterion to judge the impact of metal contamination, it implies that all forms of metals have an equal impact on the environment. Soluble, exchangeable and chelated species of metals are the most mobile components in soil, facilitating their migration and availability to the environment (Li and Thornton, 2001). Hence the efficiency of plants in influencing mobile/immobile partitioning of metals is important in soil remediation, in addition to lowering the total soil metal concentrations.  5.3.4 Relationship between soil pH and metal fractions pH is the most important factor controlling the distribution of metals among different forms, and it has the largest influence on the bio-availability of heavy metals as a result of its strong influence on solubility and speciation of metals in the soil. The relationships between soil pH and various metal fractions at 120 DAS are given in Figure 5.3. Soil pHs for five treatments – LB0, FB0, HB0, PB0 and BrB0 – were correlated with the soil metal fractions (exchangeable, oxide, organic and residual). The pH was the same (5.4) for FB0 and PB0 at 120 DAS as shown in Figure 5.3. The relationship of soil pH to metal fractions shows the differential behaviour of 97  metals to soil pH. It is seen that the exchangeable (mobile) fraction of Cu, Pb, Mn and Zn gave negative correlations with the pH of the soil, significant (P<0.05) in the case of Cu and Zn (R = 0.620 and -0.741). This reveals that mobile metal fractions (exchangeable) decrease with increase in soil pH. Each unit increase in pH results in approximately a two-fold decrease in the soluble concentration of Cu and Zn (Sanders et al. 1986). Oxide fractions of Pb and Zn gave  20  (a)  15 10 5 0 5.0  Mn fractions (mg/kg)  Pb fractions (mg/kg)  25  5.2  5.4  5.6  5.8  120 100  (c)  80  50 40  (b)  30 20 10 0 5.0  Zn fractions (mg/kg)  Cu fractions (mg/kg)  significant positive correlations (P<0.05) with soil pH (R = 0.762 and 0.792).  60 40 20 0  5.2  5.4  5.6  5.8  25 20  (d)  15 10 5 0  5.0  5.2  5.4  5.6  5.8  5.0  5.2  Soil pH  Exch  Cu Pb Mn Zn  5.4  5.6  5.8  Soil pH  Exchangeable -0.620* -0.441 -0.539 -0.741*  Oxide  Organic  Oxide -0.340 0.762* -0.289 0.792*  Residual  Organic -0.014 0.594* 0.667* -0.657*  Residual 0.172 0.076 0.136 -0.230  *Correlation coefficient was statistically significant at P 0.05. Figure 5.3. Effect of soil pH on soil metal fractions (exchangeable, oxide, organic and residual) at 120 DAS. (a) soil pH and Cu fractions, (b) soil pH and Pb fractions, (c) soil pH and Mn fractions, (d) soil pH and Zn fractions. Correlations between organic fractions of Pb and Mn and soil pH are also positive and significant (R = 0.594 and 0.667). Thus the relative partitioning of metals to the more immobile 98  forms in Festuca-growing soils for Cu, Poa-growing soils for Pb and Zn and Lolium-growing soils for Mn, with extension of plant growth from 90 to 120 DAS can be explained by the increase in soil pH over this period (Figure 5.2). An increase in exchangeable Mn, with increase in soil pH from 5.2 to 5.7 can be seen in Figure 5.3. This may be because Mn can occur in more than one valence state and the oxidized state precipitates by the formation of hydroxides (or hydrous oxides). Up to pH 6.0, the hydroxides will not precipitate and the solubility of Mn increases (Brady and Weil, 1996). 5.3.5 Relationship of plant metal concentrations to soil metal fractions  Chapter 4 examined the metal concentrations (mg/kg dry weight) and metal uptake (µg/pot) of all five studied plants. The present study further explores the relationship between metal concentrations in plants and their fractions in soil. Although correlations were found between all studied metal fractions and plant metal concentrations (both root and shoot), only the most significant correlations are presented in Figure 5.4. This figure shows how the soil metal fractionation contributes to plant absorption and, thereby, exposure to the surrounding environment. Differential behaviour of metals in influencing root and shoot absorption is evident from Figure 5.4. In the case of Cu, oxide and organic Cu contribute to plant absorption, whereas for the other three metals (Pb, Mn and Zn), exchangeable and oxide fractions contribute to plant absorption. The strengths of the correlations reveal the extent of contribution of each soil metal fraction to plant metal concentrations. Significant positive correlations (P < 0.05) were observed between oxide Cu and root Cu (R = 0.774), organic Cu and root Cu (R = 0. 619), exchangeable Pb and root Pb (R = 0. 490), oxide Pb and root Pb (R = 0.652), exchangeable Mn and root Mn (R = 0.807), oxide Mn and shoot Mn (R = 0.898), exchangeable Zn and root Zn (R = 0.822) and oxide Zn and shoot Zn (R = 0.941). These relationships indicate the mobility or bioavailability of the respective metal fractions in the soil. The results suggest that the exchangeable and oxidebound metals are of greater environmental importance than the organic and residual forms, because of their positive and significant correlations with plant metal concentrations. However, organic Cu has a significant relationship with plant Cu concentration, indicating the instability of organic Cu complexes formed at the acidic pH of the soil utilized in this study.  99  Figure 5.4 Relationship between plant metal concentrations (in root and shoot) and soil metal fractions. (a1) root Cu and oxide Cu; (a2) root Cu and organic Cu; (b1) root Pb and exchangeable Pb; (b2) root Pb and oxide Pb; (c1) root Mn and exchangeable Mn; (c2) shoot Mn and oxide Mn; (d1) root Zn and exchangeable Zn; (d2) shoot Zn and oxide Zn.  100  110  100  (a1)  90 80  Root Cu (mg/kg)  Root Cu (mg/kg)  110  70 60 50 40  100  (a2)  90 80 70 60 50 40  30 5  10  15  20  25  5  30  10  15  30  90  (b1)  80  Root Pb (mg/kg)  Root Pb (mg/kg)  90  70 60 50 40  (b2)  80 70 60 50 40 30  30 1  2  3  4  5  6  25  7  35  45  55  Oxide Pb (mg/kg)  Exchangeable Pb (mg/kg) 1000  1100  Shoot Mn (mg/kg)  Root Mn (mg/kg)  25  Oxide Cu (mg/kg)  Organic Cu (mg/kg)  (c1)  800 600 400  (c2)  900 700 500 300 100  200 5  10  15  20  25  75  30  100  125  150  175  Oxide Mn (mg/kg)  Exchangeable Mn (mg/kg) 800  Shoot Zn (mg/kg)  800  Root Zn (mg/kg)  20  (d1)  600 400 200  (d2)  600 400 200 0  0 0  5  10  15  20  25  10  20  Exchangeable Zn (mg/kg)  B0 - 90 DAS  B0 - 120 DAS  30  40  50  Oxide Zn (mg/kg)  BA - 90 DAS  BA - 120 DAS  101  Correlation Coefficient (R)  Relationship  Regression Equation  Oxide Cu vs Root Cu  y=34.508Ln(x) – 27.186  0.774*  Organic Cu vs Root Cu  y=31.202Ln(x) – 19.608  0.620*  Exchangeable Pb vs Root Pb  y=12.53Ln(x) + 45.203  0.490  Oxide Pb vs Root Pb  y=31.352Ln(x) – 52.021  0.652*  Exchangeable Mn vs Root Mn  y=375.33Ln(x) – 423  0.887*  Oxide Mn vs Shoot Mn  y=1072.2Ln(x) – 4710.5  0.898*  Exchangeable Zn vs Root Zn  y=200.35Ln(x) – 148.55  0.822*  Oxide Zn vs Shoot Zn  y=459.78Ln(x) – 1164.2  0.941*  *Correlation coefficient was statistically significant at P 0.05 There are various reaction rates that control the partitioning of metals among various soil forms, such as exchangeable, oxide, organic and residual (Adriano et al., 2004). The quantity of metal associated with a particular fraction in the soil depends on the amount of metals added, the duration of the addition, soil pH, % organic matter, clay content and soil moisture content. Plant growth plays a major role in orchestrating these factors directly by their chemical and physiological effects and indirectly by the rhizobacteria associated with them (Marschner, 1995). Because, the distribution and association of metals with various soil fractions have a direct effect on mobility and bioavailability, continuous monitoring of metal partitioning during different plant growth stages is essential for successful phytoremediation. Significant partitioning of metal fractions to insoluble forms was achieved by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. Hence Lolium, Poa and Festuca can be identified as potential phytostabilisation agents for metal-contaminants (Cu, Pb, Mn and Zn).  102  5.4 Conclusions and recommendations  • Soil pH increased from 90 to 120 days after sowing while electrical conductivity decreased over the same period. • There was a decrease in the exchangeable fraction and an increase in the oxide and organic fractions of metals in soils as a result of plant growth. Oxide fraction of metals dominated in Festuca growing soils, organic fraction in soils growing Lolium and Poa and exchangeable fraction in soil growing Helianthus and Brassica. • Exchangeable fractions of Cu, Pb, Mn and Zn were significantly negatively correlated with soil pH. Oxide fractions of Cu and Pb, and oxide as well as exchangeable fractions of Mn and Zn gave positive and significant correlations with plant metal concentrations, revealing the environmental importance of these metal fractions. • A significant partitioning of metals to insoluble forms was observed by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. Metals partitioned more to insoluble forms with the prolongation of plant growth from 90 to 120 DAS. • Lolium, Poa and Festuca appear to be suitable for phytostabilisation of Cu, Pb, Mn and Zn in moderately-contaminated acidic soils. This study assists in understanding the effects of plant growth on metal dynamics (distribution of metal fractions) in soils with variable multi-metal contamination levels at two different stages of plant growth. Since the distribution and association of metals with various soil fractions directly affect mobility and bioavailability, continuous monitoring of metal partitioning during different plant growth stages is essential to prevent associated risks. Addition of metal-specific natural soil amendments that can modify the pedogenic processes and influence the plants to immobilise metals is recommended for future study.  103  5.5 References Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142. Alvarez, E. A., Mochon, M. C., Sanchez, J. C. J. and Rodriguez, M. T. (2002). Heavy metal extractable forms in sludge from wastewater treatment plants. Chemosphere, 47, 765–775. Balasoiu, C. F., Zagury, G. J. and. Deschenes, L. (2001). Partitioning and speciation of chromium, copper and arsenic in CCA-contaminated soils: influence of soil composition. Science of The Total Environment, 280, 239–255. 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Phytoremediation: a novel strategy for the removal of toxic metal from the environment using plants. Biotechnology, 13, 468–474. Sanders, J. R., McGrath, S. P. and Adams, T. M. (1986). Zinc, Copper and Nickel concentration in rye grass grown on sludge- contaminated soils of different pH. Journal of the Science of Food and Agriculture, 37, 961-968. Sansalone, J. J., Buchberger, S. G., and Al-Abed, S. R. (1996). Fractionation of heavy metals in pavement run off. The Sci. Total Environ., 189/190, 371–378. Shuman, L. M. (1985). Fractionation method for soil microelements. Soil Sci., 140, 11-22. Singh, P and Cameotra, S. S (2004). Enhancement of metal bioremediation by use of microbial surfactants. Biochemical and Biophysical Research Communications, 319, 291–297. Singh, S., Sinha, S., Saxena, R., Pandey, K. and Bhatt, K. (2004). Translocation of metals and its effects in the tomato plants grown on various amendments of tannery wastes: evidence for involvement of antioxidants. Chemosphere, 57, 91–99. Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612. Smoley, C. K. (1992). Methods for the determination of metals in environmental samples. U.S. Environmental Protection Agency. Cincinnati, Ohio. 106  Su, D. C. and Wong, J. W. C. (2003). Chemical speciation and phytoavailability of Zn, Cu, Ni and Cd in soil amended with fly-ash stabilized sewage sludge. Environ. Int., 29, 895–900. Sutherland, R. A., Day, J. P. and Bussen, J. O. (2003). Lead concentrations, isotope ratios and source apportionment in road deposited sediments, Honolulu, Oahu, Hawaii. Water, Air, and Soil Pollution, 142, 165-186. Tessier, A., Cambell, P. G. C. and Bisson, M. (1979). Sequential extraction procedures for the speciation of particulate trace metals. Anal. Chim., 51, 844–851. Uren, N. C. and Reisenauer, H. M. (1998). The role of root exudates in nutrient acquisition. Adv. Plant Nutr., 3, 79–114. Wenzel, W. W., Lombi, E. and Adriano, D. C. (1999). Biogeochemical processes in the rhizosphere: role in phytoremediation of metal-polluted sites. In: Heavy metal stress in plants – From molecules to ecosystems. M.N.V. Prasad and J. Hagemeyer (eds) SpringerVerlag, Heidelberg, Berlin, New York, pp. 273-303. Zoubeir, L., Adeline, S., Laurent, C. S., Yoann, C., Truc, H. T., Benoît, L. G. and Federico, A. (2007). The use of the Novosol Process for the treatment of polluted marine sediment. Journal of Hazardous Materials, 148 (3), 606-612.  107  6.  5  EFFECT  OF  AMENDMENTS  ON  PHYTOAVAILABILITY  AND  FRACTIONATION OF COPPER AND ZINC IN CONTAMINATED SOIL  6.1 Introduction Copper (Cu) and zinc (Zn) are essential elements for human health, as components of many metalloenzymes and respiratory pigments. They play an important role in mammalian cellular metabolism (Buch et al., 2008). Their homeostasis in plant cells at excess levels has been reported by Palmer and Guerinot (2009). High concentrations of these metals originate mainly from mining (Prasad and Freitas, 2003). Other anthropogenic sources include industries, agriculture (via fertilizers and pesticides), incineration of wastes, vehicular traffic, as well as dressings of sewage sludge and pig slurries (Ahluwalia and Goyal, 2007; Varrica et al., 2003; Vargoa et al., 2005). They can cause long-term risks to ecosystems and threaten human health. In human beings, excess Cu causes epigastric pain, gastrointestinal effects and hemolytic anaemia (Turnlund, 1999), whereas excess Zn causes tachycardia, vascular shock, dyspeptic nausea, pancreatictis and damage of hepatic parenchyma (Barone et al., 1998; Salgueiro et al., 2000). In plants, excess copper causes toxic effects by catalyzing the production of highly toxic hydroxyl radicals from intracellularly generated hydrogen peroxide and affecting membrane properties (Prasad et al., 2001), whereas excess Zn results in stunting of roots, lignification of epidermal cells and increased permeability of root membranes (Pahlsson, 1989). Many technologies have been developed to treat and remediate Cu- and Zn-contaminated soils (Cao et al., 2003; Peng et al., 2009). Phytoremediation is a cost-effective, environmentallyfriendly technology, which could maintain the biological and functional integrity of soil after remediation (Pilon-Smits, 2005; Padmavathiamma and Li, 2007). Reducing the mobility and phyto-availability of contaminants (e.g. trace elements), through adsorption, absorption and accumulation in roots and precipitation in the rhizosphere, by growing metal-tolerant plants is termed phytostabilisation (Smith and Bradshaw, 1979). This technology is often associated with soil amendment treatments for in situ stabilisation of contaminants - “aided phytostabilisation”  5  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2009) Phytostabilisation – a sustainable remediation for zinc toxicity in soils. Water Air Soil Pollution, 9(3-4), 253-260. 108  (Bes and Mench, 2008). Some amendments such as compost, cyclonic ash, zerovalent iron grit, and phosphate, as well as plants such as Festuca rubra and Agrostis capillaris have been reported to be effective in stabilising metal-contaminants (Smith and Bradshaw, 1979; Mench et al., 2006; Brown et al., 2004; Adriano et al., 2004; Simon, 2005; Kumpiene et al., 2007; Hartley and Lepp, 2008; Bes and Mench, 2008). Few studies have been conducted on the phytostabilisation of metal-contaminated sites, metal bioavailability and metal bioaccessibility to animals (Mench et al. 2006; Brown et al. 2004). However limited comprehensive studies, using plants and natural agricultural amendments to identify an integrated, sustainable, eco-friendly and cost-effective package for phytostabilisation, have been performed in acid soils of southwest British Columbia. The present study was undertaken in a highway soil contaminated with Cu, Pb, Mn and Zn using natural agricultural amendments such as compost, lime and phosphate individually and in combination, together with three previously-identified phytoremediating plant species, Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass) (Padmavathiamma and Li, 2009a). The soils were spiked with multimetals to simulate an actual contaminated site. This chapter focuses on Cu and Zn only; whereas the next chapter considers Pb and Mn. The objectives were to: (1) investigate the effect of amendment addition on Cu and Zn accumulation in plants, and (2) assess the effect of soil-amendment–plant interaction on the mobility and phyto-availability of these metals in soil. The aging effect of the spiked soil on metal-contamination is not undertaken in the present study. pH is the most important parameter determining the effect of aging on Zn partitioning in soils, and in soils with a low pH, aging has little effect on Zn bioavailability (Lock and Janssen, 2003). The main research tasks were to (1) estimate the metal concentrations and metal uptake by plants, (2) assess the translocation properties and accumulation characteristics of these metals in plants with and without amendment, (3) investigate the distribution of metal fractions in the soil with and without plant growth and amendment addition, and (4) examine the relationship between - soil metal fractions and plant metal concentrations, biometric characteristics and plant metal concentrations, soil pH and soil metal fractions, and total soil metal concentration and plant Enrichment Coefficient (EC).  109  6.2 Materials and methods A bulk soil sample of 0-15 cm top layer was collected from the backyard of Surrey Fire hall No. 5, near a major highway intersection (HW 1 with 176 street in Surrey, British Columbia). This soil, referred to hereafter as B0, was found to be contaminated with Cu (52 mg/kg), Pb (93 mg/kg), Mn (215 mg/kg) and Zn (70 mg/kg). This soil was spiked with Cu, Pb, Mn and Zn at 30, 50, 200 and 80 mg/kg, respectively to conform to the A-level British Columbia standards for contaminated sites (Ministry of Environment, B.C, 1995), resulting in total Cu, Pb, Mn, and Zn concentrations of 80, 146, 408 and 148 mg/kg, respectively (designated BA). Amendments such as lime, phosphate and compost were added to the BA soils individually and in combination. Dolomite (finely ground) was the liming material and the source of P was CaHPO4.2H2O (41% P2O5). The compost used was City of Vancouver Yard Trimming Compost (pH - 6.4; electrical conductivity - 3.2 dSm-1; C/N ratio - 21.3, Cu – 1.2 mg/kg, Zn - 42 mg/kg, Fe – 61 mg/kg and Mn - 146 mg/kg). The detailed experimental program is summarized in Table 6.1. The nomenclature used for various treatments/conditions is given in Table 6.1. The abbreviations used for different treatments in the text, tables and figures are B0 (original soil), BA (spiked soil), BAL (spiked soil + lime), BAP (spiked soil + phosphate), BAO (spiked soil + compost) and BALPO (spiked soil + lime + phosphate + compost). The details of amendment treatments are given in “Application of amendments”, Appendix C. Table 6.1 Experimental Program for soil-plant-amendment interaction Metals studied  Conditions/Treatments  Plant species  Stage of sampling  • Cu  • Original soil with multi-metal concentrations (52 mg/kg Cu, 93 mg/kg Pb, 215 mg/kg Mn and 70 mg/kg Zn) – B0 • Original soil spiked with multi-metals to give total concentrations (80 mg/kg Cu, 146 mg/kg Pb, 408 mg/kg Mn and 148 mg/kg Zn) - BA. • BA plus lime (10 tons/ha) - BAL • BA plus phosphate (135 kg P2O5/ha) - BAP • BA plus compost (10 tons/ha) - BAO • BA plus lime plus phosphate plus compost (combined application) - BALPO)  • Poa pratensis  • 90 days after sowing  • Pb • Mn • Zn  • Lolium perenne • Festuca rubra  Design – Completely Randomized Design. 18 treatments (6 conditions and 3 plant species) with 3 replications.  110  The plant species investigated were Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass). Fifty-four plastic pots of 1 kg size were used for the experiments. The weight of each pot without soil was determined before filling it with 1 kg soil. The soil in each pot was thoroughly mixed with the required quantity of amendments and the moisture content brought to the field moisture capacity (36%). Each pot was then kept for two days for equilibration and seeds sown (0.5 g/pot). An experiment with 18 treatments and three replications was conducted in a greenhouse from August 2006 to November 2006, in a completely randomized design. The summary of weather conditions during this period is given in Appendix C, Table 2. Soil and plant samples were collected at 90 DAS (days after sowing). Shoots and roots were then separated and oven dried (70°C) to constant weight. The dried tissues were weighed and ground for metals analysis. Various biometric characters such as length of root, number of branches per root, root weight, length of shoot, shoot weight and root/shoot ratios were recorded. The original soil (B0) was analysed for basic physico-chemical characteristics such as pH (water), electrical conductivity, % organic matter, available phosphorus and total metal contents (Table 3.1). The procedure of Tessier et al (1979), as modified by Preciado and Li (2006), was adopted for selective sequential extraction. Different metal fractions were: exchangeable, carbonates and oxide, organic, and residual. The plant samples were air dried and ashed by the method outlined by Lintern et al., 1997. The ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with de-ionized water. Soil and plant extracts were analysed for Cu, Pb, Mn and Zn concentrations by means of a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Quality checking and control were performed using blanks, duplicate samples and standard solutions. The statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) to compare the treatment effects on soil metal speciation, plant metal concentration, as well as metal uptake by plants. In order to assess the phytostabilisation efficiency, the Enrichment Coefficient (EC) of root (Croots/Csoil,, the ratio of root concentration to soil concentration) and shoot (Cshoots/Csoil, ratio of shoot concentration to soil concentration) and  111  Translocation Factor (TF = Cshoots/Croots, ratio of shoot concentration to root concentration) were calculated (Kumar et al., 1995). ECroot = [Metal]root/[Metal]soil ECshoot = [Metal]shoot/[Metal]soil TF  = [Metal]shoot/[Metal]root  6.3 Results and discussion The texture of the soil was sandy clay loam and its classification was Luvisolic Humoferric Podzol according to the Canadian System of Soil Classification (1998). Basic characteristics were: pH – 5.6, electrical conductivity – 0.61 dS/m, % carbon – 1.5 and available phosphorus extracted by Bray 1 – 10.4 mg/kg. Total metal concentrations in the original soil (B0) and spiked soil (BA) were: Cu – 52 and 80 mg/kg, Pb – 93 and 146mg/kg, Mn – 215 and 408 mg/kg and Zn – 70 and 148 mg/ kg, respectively. 6.3.1 Effect of soil amendments on metal concentrations and uptake in plants. The effects of various treatments on Cu and Zn concentrations in plants are portrayed in Figure 6.1.  112  b  b  Shoot a  c  80  c ab  bc  d cd  d  40 0 BA  BAL  BAP  BAO BALPO  Cu concentration (mg/kg)  160 120 80  Root  (c) c b  d  Shoot cd  c  cd  d  40  e d  0 BA  BAL  BAP  Cu concentration (mg/kg)  b bc  Root ab  Shoot a  c  80  c ab  c  d cd  40 0 BA  BAL  BAP  ab  400  BAO BALPO  Treatments  ab  d d  e  fg  200  fg  0 BA  BAL  BAP  BAO BALPO  Treatments  800 600  a b  a  (d)  Root  c  d  b  c  400  Shoot e  200  fg  fg  0 BA  BAL  BAP  BAO BALPO  Treatments  800 600  (f)  c  ab  ab  b  f  200  Root  ab  Shoot  d  e  400  f  g  0 BA  Poa  Root Shoot  c  Festuca  160 120  600  a  (b)  a  BAO BALPO  Treatments  (e)  800  Lolium  Treatments  a  Zn concentration (mg/kg)  (a)  Zn concentration (mg/kg)  120  Root  Zn concentration (mg/kg)  Cu concentration (mg/kg)  160  BAL  BAP  BAO BALPO  Treatments  Figure 6.1 Metal concentrations in plants (in root and shoot). (a) Cu concentrations in Lolium. (b) Zn concentrations in Lolium, (c) Cu concentrations in Festuca, (d) Zn concentrations in Festuca, (e) Cu concentrations in Poa, (f) Zn concentrations in Poa. BA – spiked soil, BAL spiked soil plus lime, BAP - spiked soil plus phosphate, BAO - spiked soil plus compost, BALPO - spiked soil plus lime, phosphate and compost. F significant at P<0.05 for both Cu and Zn. Application of lime (BAL) decreased the Cu and Zn concentration in the root and shoot of all the three plant species. The decrease was significant (P<0.05) in Festuca for Cu and in Poa for Zn. The increase in soil pH (Figure 6.2) by the application of lime reduced the exchangeable or phyto-available fractions of Cu and Zn (discussed under 6.3.3), and reflected in the low plant  113  tissue concentrations of Cu and Zn. The effectiveness of lime in increasing soil pH and reducing metal availability was previously reported by Kabata-Pendias and Pendias (1992).  8.0  Soil pH  7.5 7.0 6.5 6.0 5.5 5.0 B0  BA  BAL  BAP  BAO  BALPO  Treatments Lolium  Festuca  Poa  Figure 6.2 Soil pH as influenced by plant growth and amendment application.* - F significant at P<0.05. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Festuca showed the highest Cu concentration, but the amendments significantly lowered (P<0.05) the values, with the lowest values achieved with combined application of amendments. The shoot Cu concentration, which ranged from 47 to 54 mg/kg in BA soil, decreased to <30 mg/kg as a result of combined application of amendments (BALPO). Concentrations of Cu greater than 40 mg kg−1 of dry matter may induce toxicity in plants, and may also cause toxic effects in animals (eg. sheep) feeding on them (Annenkov, 1982). Compared to BA, application of phosphate (BAP) increased plant Cu in Lolium and Poa, but decreased plant Zn in all the three plant species. This may be due to the fact that although phosphate application augmented the exchangeable Zn fraction of the soil, it may have had an antagonistic effect on Zn absorption by the plants. Similar findings, where amendment addition increased the exchangeable and soluble metal fractions in soils with no significant influence on plant concentrations, were reported by Vangronsveld et al. (2000) and Simon (2005). Compost application (BAO) reduced the plant concentration of Cu, providing evidence for Cu binding with insoluble organic 114  fractions (Hsu and Lo, 2000), whereas augmentation was found for plant Zn (Figure 6.1). Combined application of amendments (BALPO) significantly decreased the concentrations of Cu and Zn in plants, with Festuca exhibiting the lowest values for Cu and Poa for Zn (Figure 6.1). With the combined application of amendments, the plant metal concentration decreased by more than 40 % for Cu and 70 % for Zn, compared to BA soil. The highest plant biomass (both root and shoot) for all three plant species was recorded with the combined application of the amendments (2.3 g for Lolium, 1.8 g for Festuca and 2.0 g for Poa). This increase in biomass may be because; favourable plant growth under ideal soil pH (Figure 6.2), created by the combined addition of amendments provides a conducive rhizospheric climate by sequestering excess metals, along with the fertilizing effect of lime and compost. Root and Shoot biomass, dry weight (DW) (g/pot) is given in Appendix C, Table 3. The Cu and Zn uptake by plants are given in Figure 6.3. Even though the plant biomass was higher in treatments that received amendments in combination, the uptake of Cu and Zn by these plants was less than for plants that received only one amendment (Figure 6.3). The low metal uptake may be due to the low mobile or phyto-available metal fractions in the soil because of precipitation as hydroxides, phosphates or sequestration by organic ligands by combined amendment addition (BALPO). There was a significant increase in Cu and Zn uptake (P<0.05), when phosphate and compost were applied individually (Figure 6.3). This may be due to the favourable growth conditions, i.e., nutrient supply and soil physical properties, created by the application of these amendments.  115  a  a  Cu  800  Zn  600  a  a  b b  400 200  a  a  1000  c  b c.  a  a  b  b  bc c  c  ab  ab  bc bc bc ab c c c  ab  b  c  Lolium  Festuca  BALPO  BAO  BAP  BAL  BA  BALPO  BAO  BAP  BAL  BA  BALPO  BAO  BAP  BAL  0 BA  metal uptake (micro grams/pot)  1200  Poa  Figure 6.3 Metal uptake by plants. BA – Spiked soil, BAL - Spiked soil plus lime, BAP Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Mean values, n = 3. Bars followed by different letters show significantly different statistical values (P<0.05) for means. 6.3.2 Accumulation of Cu and Zn in plants as characterized by EC and TF  The Enrichment Coefficient (EC) and Translocation Factor (TF) of the three plant species after different treatments are given in Table 6.2, and the relationship between total soil metal content and EC (ECroot and ECshoot) in plants is given in Figure 6.4. Means followed by different letters show significantly different statistical values (P<0.05). For eg. ECroot of BALPO in Lolium growing soil is significantly lower than BA, BAP and BAO while it is on par with BAL. Compared to BA, application of lime (BAL) significantly lowered the ECroot, ECshoot and TF values of Cu and Zn (P<0.05). On the other hand, compared to BA, application of phosphates (BAP) increased the EC values (both root and shoot) of Cu in all the three plant species, not significant though. Compost application (BAO) had an augmenting effect only on ECroot of Zn (5.90) in Poa (Table 6.2). The lowest ECroot, ECshoot and TF for Cu (0.70, 0.36 and 0.45, respectively) were by Festuca, and for Zn (0.97, 0.63 and 0.44, respectively) by Poa, in both cases with combined application of amendments (BALPO). Low EC and TF values reveal a low plant absorption as well as translocation of metals in the plants. The differences between plant species in metal accumulation and translocation may be due to different release of root exudates, e.g. organic acids, CO2 and H+ that can change soil pH and control the release or sequestration of metals (Kelly et al., 1998). The ionic competition, high soil pH and binding of metals by root exudates may be the reasons for the lower translocation of metals to the above ground parts of 116  plants in amendment-applied treatments (Kumpiene et al., 2007). Also indirect application of Ca through lime may enhance the Ca/metal competition and a higher cellular Ca which may contribute to membrane integrity, lowering the oxidative stress, for maintaining the cell homeostasis (Prasad, 2004). Table 6.2 Enrichment Coefficient (EC) and Translocation Factor (TF) in different treatments by plant growth and amendment additions. Plant Enrichment Co-efficient Translocation Factor (TF) species Cu Zn  Lolium  Festuca  Treatments  ECroot  ECshoot  ECroot  ECshoot  Cu  Zn  B0 BA BAL BAP BAO BALPO  1.50a 1.19b 0.87cd 1.25b 0.95c 0.78d  0.71c 0.64cd 0.48e 0.69c 0.67cd 0.48e  2.44d 4.25b 2.15de 2.88cd 3.87bc 1.41ef  2.35b 3.55ab 0.83c 2.44b 3.79ab 0.73c  0.48d 0.54cd 0.55cd 0.54c 0.71a 0.62bc  0.96a 0.84bc 0.39ef 0.84bc 0.97a 0.76c  B0 BA BAL BAP BAO BALPO  1.39ab 1.41a 0.73d 1.47a 1.21b 0.70d  0.94a 0.67cd 0.45ef 0.65cd 0.67cd 0.36f  3.29c 4.74ab 2.58cd 5.39a 5.01a 1.12ef  3.11b 4.03a 0.84c 3.76ab 3.99a 0.74c  0.68ab 0.48d 0.56c 0.47d 0.56c 0.45d  0.95a 0.85b 0.33f 0.69cd 0.60d 0.66c  B0 0.85cd 0.63cd 2.79cd 2.65b 0.74a 0.95a BA 1.22b 0.58d 4.87b 4.33a 0.48d 0.89ab Poa BAL 0.72d 0.41ef 1.96e 1.09c 0.52cd 0.59d BAP 1.36ab 0.87ab 4.57ab 4.24a 0.64b 0.93a BAO 1.10bc 0.80bc 5.90a 2.63b 0.73a 0.45e BALPO 0.91c 0.63cd 0.97f 0.63c 0.69ab 0.44e F * * * * * * Mean values, n = 3. *F significant at P<0.05. Significantly different statistical values (P<0.05) according to the Least Significance Test in each column are followed by different letters. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. The relationship between total soil metal concentration and ECroot and ECshoot (Figue 6.4) reveals that as the total metal concentration in the soil increased, ECroot and ECshoot decreased for both Cu and Zn. ECroot and ECshoot for Cu and Zn were significantly correlated (P <0.05) with total 117  metal concentrations in the soil (R values being 0.882 and 0.807 for Cu, and 0.674 and 0.811 for Zn, respectively).  Enrichment Coefficient  1.6  y = -3.2766Ln(x) + 15.436 R = 0.882  1.2  0.8  0.4  y = -1.8445Ln(x) + 8.7094 R = 0.807  0 70  75  80  85  90  Total Cu in the soil (mg/kg) EC root  EC shoot  Enrichment Coefficient  8  y = -8.5426Ln(x) + 44.645 R = 0.674  6  4  2  y = -8.5378Ln(x) + 43.498 R = 0.811  0 85  95  105  115  125  135  145  Total Zn in the soil (mg/kg) EC root  EC shoot  Figure 6.4 Relationship between soil metal concentrations and Enrichment Coefficients (EC) for root and shoot Such an accumulation pattern, when the higher soil metal concentrations do not cause higher metal accumulation by plants, is favourable for phytostabilisation. This can be explained by the plant’s inherent ability to immobilise metals and decrease its absorption when the soil metal concentrations are high (Salisbury and Ross, 1992). The immobilization of metals in polluted 118  soils by the growth of grasses has been reported by Bogatu et al. (2007). On the contrary, there are reports of increased metal uptake under low levels of soil metals, when plant roots increase metal bioavailability by extruding protons, phytosiderophores, and organic acids, acidifying the soil and mobilising metals (Marschener, 1998; Garbisu and Alkorta, 2001). Grasses are known to excrete phytosiderophores and LMWOA (low-molecular-weight organic acids), which are chelating agents, that bind metals into metal-chelate complexes, making them more available for plant uptake at low concentrations of soil metals (Ma and Nomoto, 1996).  6.3.3 Metal fractionation in the soil  Cu and Zn fractionation in the soil due to plant growth alone is presented Table 6.3, and by plant growth and amendment addition is given in Figure 6.5. It is seen that when the soil was spiked, there was an increase in the oxide fraction for Cu, whereas for Zn, the increase was mainly in the exchangeable fraction (Table 6.3). Growth of Lolium enhanced the exchangeable Cu fraction, whereas Festuca had a lowering effect (Table 6.3), in agreement with results from a previous study (Padmavathiamma and Li, 2009b). When compared to BA, lime application (BAL) significantly reduced exchangeable fractions of Cu and Zn (P<0.05), with this reduction being highest in Festuca-growing soils for Cu and Poa-growing soils for Zn (Figure 6.5). Similar observations on the effect of lime in reducing the mobile metal fractions were reported by Hooda and Alloway (1996). Lime amendments increase soil pH (McBride et al., 1997) and favour the formation of oxides, metal-carbonate precipitates, and other complexes that decrease metal solubility and bio-availability (Mench et al., 2006). Application of phosphates (BAP) significantly increased the exchangeable fraction in soils growing Poa and Lolium for Cu and Lolium and Festuca for Zn (Figure 6.5). A decrease in exchangeable Zn by phosphate application (BAP) when compared to BA, in Poa growing soils was noticed. This may be due to the immobilization of Zn as Zn phosphates, with low solubility and high resistance to soil acidification (Impellitteri, 2005). There was a significant increase in the organic fraction of Cu with the addition of compost (BAO), the highest value being recorded by soils growing Poa (Figure 6.5). The organic-bound Cu fraction was found to account for 50% of the total soil Cu in the Poa grown soil with combined amendments (BALPO). This may be due to a greater number of fine roots when plant growth is promoted by addition of compost. The number of branches/roots recorded in Poa (18) was significantly higher compared to that in Lolium (12) and Festuca (10.3) in BAO. This increase in organic-bound Cu fraction in Poa grown soil may 119  reflect Cu storage in fine roots and root hairs. However for Zn, the amount partitioned in the organic fraction was less, even in compost-applied treatments. Organic amendments can contribute to metal immobilisation through the formation of stable complexes with OH or COOH groups on the solid surfaces of the organic polymers (Chirenje and Ma, 1999). However, this may promote metal mobility if complexes formed with the amendments are more soluble than without them (Hsu and Lo, 2000). Table 6.3. % partitioning of Cu and Zn in soils with and without plant growth.  % Cu fractions in soil  Treatments /Conditions  Exch. Oxide Soil alone  Soil + Lolium  Soil + Festuca  Soil + Poa  Organic  % Zn fractions in soil  Residual Exch. Oxide  Organic  Residual  B0  4.4  22.1  33.5  40  11.9  43.5  18.5  26.1  BA  4.9  25.8  31  38.3  21.8  39.1  11.3  27.7  B0  9.4  16.7  34.1  39.8  12.2  25.6  23.3  38.9  BA  6.1  30.1  24.7  39.1  22.8  30.6  15.7  30.8  B0  2.3  50.7  20.3  26.7  10.4  40.9  18.2  30.8  BA  2.1  43.6  17.6  36.7  24.1  41.7  13.1  20.7  B0  5.9  15.6  35.6  42.8  4.1  40.2  18.4  36.9  BA  4.7  24.1  40.5  31  8.5  45.1  13.9  32.9  The major partitioning of each metal in a soil is in bold. n=3. F significant at P<0.05. B0 – Initial soil, BA – Spiked soil.  120  % Cu fractionation  100% 80% 60% 40% 20%  Amendments alone  Amendments + Lolium  Amendments + Festuca  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  0%  Amendments + Poa  % Zn fractionation  100% 80% 60% 40% 20%  Amendments alone  Exch  Oxide  Organic  Amendments + Lolium  Amendments + Festuca  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  0%  Amendments + Poa  Residual  Figure 6.5 Partitioning of Cu and Zn in soil by the effect of amendments and plants. BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Mean values, n = 3. F significant at P<0.05. Among the different treatments, maximum immobilisation of Cu and Zn in the soil was achieved by the combined application of amendments (BALPO) with Festuca and Poa, respectively. Combining amendments can be beneficial for maintaining a favourable soil pH (Jackson and Miller, 2000) and increasing Cu sorption on clay mineral surfaces through ternary Cu-mineralhumic acids complexes (Hizal and Apak, 2006). Also, with the combined application, the Ca content of lime can bind to phosphorus, making it less available to plants and thereby reducing its influence on increasing the mobile metal fraction (Schnoor, 1996). In the present study, since  121  lime and compost were added in conjunction with phosphate, the lowering of pH by phosphate was nullified by lime, creating a favourable environment for metal immobilisation in the soil.  6.3.4 Relationship between soil pH and metal fractions  Lime application (BAL) increased soil pH from 5.4 to from 7.2; whereas phosphate addition (BAP) decreased the pH in Festuca growing soils from 5.4 to 5.1. Compost application (BAO) had also an augmenting effect on soil pH. The relationship between soil pH and Cu fractions in soil indicate that soil pH had a significant influence on the exchangeable and organic Cu fractions (Figure 6.6). Exchangeable Cu was significantly negatively correlated with pH (Table 6.4) in soils growing all three plant species, the highest correlation being for Lolium (R = 0.976). The organic fraction of Cu in Poa growing soils showed a significant positive correlation with soil pH (R = 0.663). As in the case of Cu, exchangeable Zn was also significantly and negatively correlated with pH, the greatest correlation coefficient being for soils growing Lolium (R = -0.802). Other Zn fractions in the soil (oxide, organic and residual) were positively and significantly correlated with pH (Table 6.4) in Poa growing soils (R values being 0.902, 0.565 and 0.656 respectively).  122  Zn fractions (mg/kg)  Cu fractions (mg/kg)  40  (a)  30 20 10  80  40 20  0 5.0  5.5  6.0  6.5  7.0  5.0  Zn fractions (mg/kg)  Cu fractions (mg/kg)  20 10  6.0  6.5  7.0  80  10 0 6.5  7.5  7.0  7.5  20 0 5.5  7.0  7.5  pH of soil  Exch.  6.5  80  (f)  60 40 20  (Poa) Oxide  6.0  pH of soil  Zn fractions (mg/kg)  Cu fractions (mg/kg)  20  6.0  7.0  40  (Festuca)  (e)  5.5  7.5  (d)  5.0  40  5.0  7.0  60  7.5  pH of soil  30  6.5  100  0 5.5  6.0  (Lolium)  (c)  5.0  5.5  pH of soil  40 30  0  7.5  pH of soil  (b)  60  Organic  0 5.0  5.5  6.0  6.5  pH of soil  Residual  Figure 6.6 Relationship between soil pH and metal fractions. (a) soil pH and Cu fractions in Lolium soil, (b) soil pH and Zn fractions in Lolium soil, (c) soil pH and Cu fractions in Festuca soil, (d) soil pH and Zn fractions in Festuca soil, (e) soil pH and Cu fractions in Poa soil, (f) soil pH and Zn fractions in Poa soil.  123  Table 6.4 Correlation coefficients between soil pH and metal fractions Cu  Zn  Lolium  Festuca  Poa  Lolium  Festuca  Poa  Exchangeable  -0.976*  -0.835*  -0.609*  -0.802*  -0.789*  -0.781*  Oxide  0.135  0.465  0.349  0.878*  0.716*  0.902*  Organic  0.424  0.447  0.663*  0.417  0.355  0.565*  Residual  0.445  0.581*  0.301  0.600*  0.647*  0.656*  *Correlation coefficient was statistically significant at P<0.05 Thus, stability of Cu and Zn in soil was strongly pH dependent – the mobility increasing with decreasing pH (Kabata-Pendias and Pendias, 1992). Also, a significant influence of pH was observed on the organic fraction of Cu and on the oxide and exchangeable fractions of Zn (Figure 6.6). Growth of plants alter the pH conditions of the soil by releasing of organic acids, generating CO2 from root respiration, exuding protons, and by the imbalance in cation or anion release caused by excess anion or cation uptake (Kelly et al., 1998). Because speciation varies with pH, the metal ion concentration exerting a given toxic effect can be expressed as a function of pH (Lofts et al., 2004). Predicting pCu2+ (soil-solution free copper activity) as a function of total soil copper and soil pH is a better exposure indicator (Sauve et al., 1997).  6.3.5 Relationship of plant metal concentration to soil metal fractions  Correlations between plant metal concentrations and soil metal fractions are given in Table 6.5. Both root and shoot Cu concentrations were positively correlated with exchangeable Cu (R of 0.661 and 0.589, respectively) and oxide Cu (R of 0.750 and 0.634, respectively) in soil (Table 6.5). Even though organic-bound Cu in soil was higher than other fractions, it did not influence the plant Cu concentration, revealing the non-availability of Cu-organic complexes to plants. In the case of Zn, a significant positive correlation was observed between exchangeable Zn fraction in the soil and both root and shoot Zn (Table 6.5). A lack of correlation between oxide-bound Zn in the soil and plant Zn concentration indicated the unavailability of that soil fraction to the plants, like Cu-organic complexes.  124  Table 6.5.  Correlations between plant (root and shoot) metal concentrations and soil metal fractions.  Soil metal fractions  Cu shoot  Cu root Regression equation  R  Regression equation  R  Cu exch.  y = 8.98 x + 52.1  0.661*  y = 5.16 x + 33.8  0.589*  Cu oxide  y = 2.62 x + 28.2  0.750*  y = 1.43 x + 21.5  0.634*  Cu organic  y = 0.11 x + 73.7  0.038  y = 0.12 x + 44.9  0.061  Cu residual  y = 0.52 x + 64.9  0.132  y = -0.71x + 63.3  -0.277  Zn shoot  Zn root Regression equation  R  Regression equation  R  Zn exch.  y = 17.5 x + 180  0.890*  y = 12.3 x + 118  0.986*  Zn oxide  y = -1.93 x + 52  -0.164  y = -3.73 x + 479  -0.448  Zn organic  y = -2.3 x + 470  -0.083  y = -8.9 x + 468  -0.450  Zn residual  y = 2.51 x + 327  0.115  y = -0.63 x + 316  -0.040  *Correlation coefficient (R) was statistically significant at P<0.05.  6.3.6 Plant metal concentrations and biometric characteristics  The correlation coefficients between biometric characteristics and plant concentrations (Cu and Zn) appear in Table 6.6. In Poa and Lolium, shoot length had a positive correlation with plant Cu (both root and shoot), whereas root length was negatively correlated with plant Cu and Zn. In Festuca, the number of root branches was significantly negatively correlated with plant Cu (root and shoot). In the case of plant Zn, none of the biometric characters was found to have a pronounced influence in Festuca, whereas in Poa, Zn  root  was positively and significantly  correlated with shoot length (R = 0.506), and Zn shoot was significantly negatively correlated with root length. (R = -0.556). Thus, a negative relationship was shown by root parameters such as root length and number of root branches on metal concentration in plants. Exudates released by plant roots can bind with metals transforming them into organo-metallic complexes, which are neither mobile nor bio-available. These are important aspects of phytostabilisation, since the efficiency of rhizosphere metal precipitation depends on the exposure of plant roots to the contaminated zones. 125  Table 6.6.  Correlation coefficients concentrations.  Plant species  Lolium  Festuca  Poa  between  plant  biometric  characters  and  metal  Metal concentration  Shoot length  Root length  Number of branches/root  Cu root Cu shoot Zn root Zn shoot Cu root Cu shoot Zn root Zn shoot Cu root Cu shoot Zn root Zn shoot  0.891* 0.776* 0.499 0.332 -0.338 -0.287 0.036 -0.423 0.699* 0.682* 0.506* 0.208  -0.889* -0.778* -0.659* -0.686* -0.556* -0.399 -0.094 -0.124 -0.254 -0.2359 0.100 -0.556*  -0.091 0.005 0.232 -0.083 -0.766* -0.825* -0.353 -0.216 0.362 0.315 0.364 0.232  *Correlation coefficient was statistically significant at P 0.05. Root parameters contribute to rhizosphere metal dynamics and ultimately the effectiveness of plants for soil metal de-contamination (Uren and Reisenauer, 1998; Marschner, 1998). The amount and composition of root exudates released into the rhizosphere are highly variable and dependent on plant species, stage of plant growth, physico-chemical environment and metal toxicity stress (Marschner, 1998).  6.4 Conclusions and recommendations  The results demonstrate the effectiveness of soil-plant-amendment interaction in suppressing the availability of Cu and Zn in a contaminated acid soil. Specific findings were:  •  Changes in soil pH due to the application of lime had a significant effect on the exchangeable fractions of Cu and Zn, organic fractions of Cu, and oxide fractions of Zn in soil. Addition of lime to the soil lowered the plant Cu and Zn concentrations.  126  •  Phosphate application increased the exchangeable Cu content in soil, increased plant Cu, and decreased plant Zn.  •  Application of compost significantly increased the organic fraction of Cu, especially in soils grown with Poa, while, no pronounced effect on Zn fractionation was observed.  •  With combined amendments, the concentration of Cu in shoots and roots decreased by more than 40 % in Festuca, and by ~70 % for Zn in Poa.  •  Maximum metal immobilisation was achieved in the soil by the combined application of all amendments, in conjunction with growth of Festuca for Cu and Poa for Zn.  •  `Lowest EC and TF values were observed in Festuca for Cu and Poa for Zn with combined application of amendments.  Since the soil contained multiple metal contaminations, the different properties of the contaminants restrict the choice of possible amendments in order to avoid large pH fluctuations and consequent mobilization of one or more of the metals. The presence of Cu can decrease the stabilization efficiency of Zn due to competition for sorption sites. Thus the treatment efficiency of multi-element contaminated sites can be improved only by applying a combination of amendments. In such cases, lowering of soil pH due to phosphate can be countered by adding lime. However, there is need to confirm these results under field conditions. The metal speciation due to aging in the spiked soil, the functioning of root metal transporters and the change in microbial biomass and mycorhization are suggested as future studies.  127  6.5 References  Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121-142. 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Vargova, M., Ondrasovicova, O., Sasakova, N., Ondrasovic, M., Culenova, K and Smirjakova, S. (2005). Heavy metals in sewage sludge and pig slurry solids and the health and environmental risk associated with their application to agricultural soil. Folia Veterinaria, 49, 28-30. Varrica, D., Dongarra, G., Sabatino, G. and Monna, F. (2003). Inorganic Geochemistry of Roadway Dust from the Metropolitan Area of Palermo, Italy. Environmental Geology, 44, 222-230.  131  7.  6  PHYTOAVAILABILITY AND FRACTIONATION OF LEAD AND MANGANESE IN CONTAMINATED SOIL FOLLOWING APPLICATION OF THREE AMENDMENTS  7.1 Introduction  Pb and Mn are the two metals that have been used extensively in transport vehicles during the past few decades. The use of tetraethyl lead (TEL) as an antiknock compound for gasoline engines in the early 1970s and the subsequent replacement by methyl cyclopentadienyl manganese tricarbonyl (MMT), have led to considerable exhaust emissions of Pb and Mn. Both are released to the environment by other anthropogenic activities too. For example, Pb contamination results from mining and smelting activities, use of Pb in paints as well as from the disposal of municipal sewage sludge and industrial wastes enriched in Pb (Joint FAO/WHO Expert Committee on Food Additives, 2000; Ma et al., 1995). Because of the high binding strength of Pb to soil fractions, Pb is highly immobile in soil and it becomes virtually permanent, with a soil retention time of 150 to 5000 years (Friedland, 1990). Hence, even though the use of leaded gasoline was suspended several decades ago in North America and most of the industrialized world, many roadside soils remain contaminated with Pb (Sezgin et al., 2003). In Canada, the anthropogenic emissions of Mn amounted to 1225 tons, with approximately 75% from industrial facilities and 20% from gasoline-powered motor vehicles using MMT (Environment Canada, 1987). Of particular concern is the effect of transportation systems on the release of these metals to the surrounding environment. Contamination of soil by lead is of major concern due to its high toxicity to humans and animals and its bioavailability through ingestion or inhalation. Relatively low concentrations of Pb in the blood can affect children's mental development, an effect that persists into adulthood (Needleman et al., 1990; Laidlaw et al., 2005). Exposure to high concentrations of Mn can lead to numerous health problems, including neurodegenerative disorders similar to Parkinson's disease (such as manganism) (US EPA, 2003). Phytoremediation is a cost-effective, environmentally friendly and ecologically sound remediation method (Baker et al., 1994) for  6  A version of this chapter has been accepted for publication. Padmavathiamma, P.K. and Li, L.Y. (2010). Phytoavailability and fractionation of lead and manganese in contaminated soil following application of three amendments. Bioresource Technology. 132  metal contaminated sites. Depending upon the conditions of the site, level of clean up required and plant species, the remediation method can be either containment or removal (Padmavathiamma and Li, 2007). Containment by in-situ immobilisation or in-place inactivation of contaminants using plants and amendments is phytostabilisation (Smith and Bradshaw, 1979; Arienzo et al., 2003). This may be suited for busy contaminated sites such as highway soils, where contaminant removal is not feasible and practical due to physical and financial constraints. Many amendments and plants have been reported to be effective for the stabilisation of different metal-contaminants (Simon, 2005; Kumpiene et al., 2007). But a holistic approach involving suitable plants and natural amendments that can remediate the metal-contaminated sites and retain the functional and ecological integrity of soil is still lacking. Hence, the present study was undertaken in contaminated acidic soils of coastal British Columbia using natural agricultural amendments such as lime, phosphate and compost individually and in combination along with the plant species: Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass). These plants were identified to be suitable for phytostabilisation from a previous study (Padmavathiamma and Li, 2009). The objectives were: (1) to assess the effect of soil-amendment–plant interaction on the partitioning of Pb and Mn into various soil fractions and to evaluate the mobility and phyto-availability of these metals in soil, and (2) to assess the effect of soil improvement or amelioration on the accumulation characteristics and translocation properties of these metals in the plants. The studies generated sufficient data to suggest a cost effective package that can not only reduce the hazard associated with the presence of excess Pb and Mn, but also improve the soil quality and sustain its functionality.  7.2 Materials and methods  The present study was performed in soil collected from the backyard of Surrey Fire hall No. 5, near the main intersection of highway 1 with 176th Street in Surrey, British Columbia. This represents a busy site with respect to traffic counts (>80,000 vehicles/day), and it is highly contaminated with Cu, Pb, Mn and Zn (Preciado and Li, 2006). This chapter focuses on Pb and Mn, whereas the previous chapter considered Cu and Zn. The metal interaction studies by  133  Padmavathiamma and Li (2009) revealed a high degree of correlation between Cu and Zn, and limited correlation between Pb and Mn. Gasoline combustion may be the main source of Pb and Mn in highway soils. The original soil (B0) containing 52 mg/kg Cu, 93 mg/kg Pb, 215 mg/kg Mn, 70 mg/kg Zn, was spiked with further Cu, Pb, Mn and Zn, resulting in total measured Cu, Pb, Mn, and Zn concentrations of 80, 146, 408 and 148 mg/kg, respectively (designated BA), approximately matching the British Columbia Ministry of Environment (1995) A-level limits for contaminated sites. Details on the soil spiking with multi-metals and addition of soil amendments are given in Chapter 6. The plant species used for the study were Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass). The study was conducted as a pot experiment in a completely randomized design with 18 treatments and three replications (Table 6.1). The experiment was done in the greenhouse during the period, August 2006 to November 2006. Soil and plant samples were collected at 90 DAS (days after sowing). Basic characteristics of the soil such as pH, electrical conductivity, organic carbon, available P and texture were estimated. The procedure of Tessier et al (1979), as modified by Preciado and Li (2006), was adopted for selective sequential extraction. The different metal fractions estimated were: exchangeable, carbonates and oxides, organic and residual. The plant samples were air dried and ashed according to method outlined by Lintern et al., 1997. The ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with de-ionized water. Soil and plant extracts were analysed for Pb and Mn using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. The statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. Correlation and regression analyses were conducted to establish the relationship between different parameters. When R was statistically significant at P ≤ 0.05, an asterisk (*) is provided to denote the statistical significance. In order to assess the efficiency of plants for phytostabilisation, the Enrichment Coefficient (EC) of root (Croots/Csoil,, the ratio of root concentration to soil concentration) and shoot (Cshoots/Csoil, ratio of shoot concentration to the soil concentration) and Translocation Factor (TF = Cshoots/Croots, ratio of shoot concentration to the root concentration) were calculated (Kumar et al., 1995). 134  7.3 Results and discussion  The chemical characteristics of the studied soils are given in Table 7.1. Table 7.1. Physico chemical properties of the studied soils  Soil  Electrical Conductivity pH (dSm-1)  % Carbon  Total N (%)  Available P (mg kg-1)  Total metal concentrations (mg kg-1) Cu  Pb  Mn  Zn  Original soil (B0)  5.6  0.61  1.50  0.15  10.4  52  93  215  70  Spiked soil (BA)  5.4  1.27  1.28  0.11  16.7  80  146  408  148  7.3.1 Metal concentrations and uptake in plants  The plant concentration (mg/kg) and plant uptake (µg/pot) of Pb and Mn (both root and shoot) are given in Table 7.2. Means followed by different letters indicated as superscripts in Table 7.2 show significantly different statistical values (P<0.05). The lowest plant concentrations, as well as uptake (both root and shoot) (significant at P<0.05), were observed in Lolium for Mn, when compared to Festuca and Poa. In the case of Pb, for B0 and BA soils, the lowest root concentration was observed in Poa (32 and 61 mg/kg, respectively), though not statistically significant. The combined application of amendments (BALPO) lowered the plant concentrations of Pb and Mn more than the individual additions of amendments. Lime amendments (BAL) reduced both plant Pb and Mn (concentration and uptake), whereas phosphate amendments (BAP) decreased the plant Pb and increased the plant Mn (concentration and uptake).  135  Table 7.2. Metal concentrations and metal uptake by the plants (root and shoot). Mn Plant species  Lolium  Festuca  Poa  Pb  Plant concentration (mg/kg)  Plant uptake (µg/pot)  Plant concentration (mg/kg)  Plant uptake (µg/pot)  Root  Shoot  Root  Shoot  Root  Shoot  Root  Shoot  B0  276g  179j  146i  174h  36bc  17ab  19c  13bc  BA  914cd  835e  475fg  592f  65a  18a  31a  12bc  BAL  787d  639fg  535f  686ef  40b  8d  27a  8cd  BAP  1887a  1170c  1460a  1474b  35bc  11c  25b  13bc  BAO  974c  1003d  749d  1404b  44b  16ab  33a  22a  BALPO  535e  506h  267h  759e  27cd  9cd  20c  11c  B0  297fg  246ij  151i  132h  41b  21a  20c  13bc  BA  1051c  1023d  294h  429g  67a  20a  18c  8cd  BAL  929cd  780ef  455g  678ef  45b  9cd  22bc  7d  BAP  1973a  1283b  1210b  1308c  34bc  10cd  20c  10c  BAO  1053c  1090cd  651e  1253c  40b  17a  22bc  19a  BALPO  861d  607g  542f  758e  30c  8d  19c  21a  B0  390f  297i  205h  175h  32bc  19a  20c  11c  BA  1691b  1766a  845cd  918d  61a  20a  32a  10c  BAL  840d  686fg  529f  434g  42b  7d  26b  4d  BAP  1985a  1209b  1207b  1414b  35bc  13b  22bc  15b  BAO  1143c  1178bc  910c  1763a  49b  15b  38a  19a  BALPO  861d  607g  542f  758e  30c  8d  19c  21a  F  *  *  *  *  *  *  *  *  Conditions/ Treatments  Mean values, n = 3. * F significant at P<0.05. Statistically significantly different values (P<0.05) according to the Least Significance Test. in each column are followed by different letters. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. The reduction in plant Pb by phosphate application may be due to the precipitation of Pb as Pb phosphate in the soil. The acidic pH of the studied soil (5.4) and the source of P, which is Ca HPO4. 2H2O (41 % P2O5) can contribute to the precipitation of Pb phosphate in the studied soil. Apart from the addition of phosphates to the soil, root exudates from plants contain phosphatase 136  enzymes that can convert organic P to phosphate in the rhizosphere (Haeussling and Marschner, 1989) and this free phosphate would be available to metal compounds to form metal phosphates. The solubility of metal phosphates formed control the bioavailability of that fraction. The combined application of amendments (BALPO) decreased the plant Pb by 55 – 68 % and plant Mn by 40-49 %, when compared to BA. The lowered metal concentration in plants may be due to the less bioavailable fraction, most likely the result of the increase in soil pH by lime application, which ionises pH-dependent exchange sites, raising cation exchange capacity (CEC) and metal sorption to soil particles (Mench et al., 2000). Also compost and phosphate additions lead to the formation of complexes and precipitates, which lower the mobile metal fractions thereby reducing the absorption by plants (Mench et al., 2000). The concentration of Pb in the shoot was three to six times lower than that of the concentration in the root, suggesting a low translocation rate. The retention of Pb in the roots is due to binding to ion exchange sites and extra cellular precipitation, mainly in the form of Pb phosphates, with both these mechanisms occurring in the cell wall (Jarvis and Leung, 2002). Also Pb does not always penetrate the root endoderm and enter the stele since the endoderm acts as a barrier to Pb absorption and penetration to the interior of the stele and its transport to the aerial plant part (Weis and Weis, 2004). Changes in rhizospheric soil properties induced by root exudates (amino acids, sugars, organic acids, peptides, proteins etc) and amendments may have significant influence on the mobility, bioavailability and translocation of trace metals (Arienzo et al., 2003).  7.3.2 Accumulation characteristics of Pb and Mn in plants  ECroot, ECshoot and TF values of Pb and Mn are given in Table 7.3. In the case of Mn, ECroot ranged from 1.4 – 5.9 whereas ECshoot values were from 1.6 – 5.4. For Pb, the corresponding values were 0.13 – 0.49 and 0.05 – 0.25 respectively. Combined application of amendments (BALPO) significantly reduced the ECroot and ECshoot of both Mn and Pb. ECroot and ECshoot for Pb decreased from 0.47 to 0.13 and 0.15 to 0.06, respectively, with the combined addition of amendments (BALPO) in Poa. In the case of Mn, application of lime (BAL) decreased the ECroot and ECshoot, whereas phosphate (BAP) and compost (BAO) application increased the corresponding values. This can be explained by the increased exchangeable Mn fraction in the soil, which received phosphate and compost amendments, in addition to differential metal absorption by plant species due to the influence of variable root exudates released into the rhizosphere. Compared to BA, ECroot and ECshoot for Mn decreased by 60 and 66 % in Poa with 137  the combined application of amendments (BALPO), whereas the corresponding decreases in Lolium were 48 and 43 %. These results again highlight the superiority of combined amendment addition (BALPO) with Poa in stabilising Pb and Lolium in stabilising Mn in the soil. Table 7.3. ECroot, ECshoot and TF for Pb and Mn  Plant species  Lolium  Festuca  Poa  Conditions/ Treatments  B0 BA BAL BAP BAO BALPO B0 BA BAL BAP BAO BALPO B0 BA BAL BAP BAO BALPO F  Mn Enrichment Coefficient (EC) Root  Shoot  1.40 2.50 2.00 5.40 2.70 1.20 1.45 3.00 2.46 5.94 3.21 2.56 2.1 5.10 2.20 5.70 3.40 1.98 *  1.61 2.26 1.75 3.23 2.79 1.00 1.87 2.93 2.06 3.84 3.06 1.66 1.80 5.40 1.66 3.60 3.80 1.70 *  Pb TF  0.94 0.91 0.87 0.59 1.03 0.80 0.67 0.97 0.84 0.65 0.95 0.65 1.61 1.04 0.76 0.64 1.12 0.88 *  Enrichment Coefficient (EC) Root  Shoot  0.42 0.44 0.29 0.25 0.32 0.19 0.49 0.50 0.33 0.24 0.29 0.28 0.37 0.47 0.30 0.26 0.37 0.13 *  0.06 0.13 0.06 0.08 0.12 0.07 0.25 0.15 0.06 0.07 0.13 0.12 0.22 0.15 0.05 0.10 0.11 0.06 *  TF  0.47 0.29 0.20 0.31 0.38 0.35 0.51 0.29 0.21 0.29 0.42 0.36 0.59 0.3 0.16 0.37 0.3 0.22 *  Mean values, n = 3. * F significant at P<0.05. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO Spiked soil plus lime, phosphate and compost.  138  The possible explanation for the superiority may be the ability of plant roots to alter soil conditions, such as pH, organic carbon and soil moisture by root exudation (Susarla et al., 2002) and the soil amendments complementing the plant effect to bring out the assisted natural remediation. The environmental hazards of metal pollution depend on geochemical and biochemical properties of a given metal and are related to several processes taking place in the soil and plants (Mench et al., 2000). The TF (Translocation Factor) for Pb ranged from 0.16 to 0.51 whereas for Mn, it ranged from 0.59 to 1.6 (Table 7.3). For Mn, the lowest TF values were for phosphate amended soils, whereas for Pb, the lowest TF values corresponded to lime amended soil. The lowering of metal transport in plants may be explained by the fact that once they enter the plant, the metals are too insoluble to move freely in the vascular system since they form carbonate or phosphate precipitates immobilizing them in apoplastic (extracellular) and symplastic (intracellular) compartments in the root (Raskin et al., 1997). Also unless the metal ion is transported as a non-cationic metal chelate, apoplastic transport is further limited by the high cation-exchange capacity of cell walls (Raskin et al., 1997).  7.3.3 Pb and Mn fractions in the soils  Pb and Mn fractionation in the soil resulting from plant growth alone is presented in Table 7.4 and by plant growth and amendment addition in Figure 7.1. When the soil was spiked, there was an increase in the exchangeable fraction of both Pb and Mn in soil. The decrease of the mobile fraction was best achieved by the growth of Poa for Pb and Lolium for Mn. Application of amendments had a pronounced effect in further lowering the exchangeable fraction. The maximum decrease was observed in Poa with combined amendments (BALPO) for Pb, and in Lolium with lime amendments (BAL) and combined amendments (BALPO) for Mn. In general, the order of amount of Pb forms in soils with combined addition of amendments (BALPO) was: Pb  residual  > Pborganic > Pboxide > Pbexchangeable. Addition of lime  (BAL) re-distributed >40 % of total Pb to the oxide fraction, whereas addition of phosphate (BAP) re-distributed >62% of total Pb to the residual fraction (mainly bound in silicates). The organic Pb partitioning by compost application was about 32 % of the total Pb in soils grown with Poa. Pb added to soil may react with available soil anions such as SO42-, H2PO41-, HPO42- or CO32- to form sparingly soluble salts and compounds such as lead carbonate Pb (OH) (CO ) and chloropyromorphite (Pb (PO ) Cl) which are least soluble at near 3  2  3 2  5  4 3  139  neutral pH (Waldron, 1980). This may be the reason for lowering the mobile Pb fraction in soils with the combined amendment addition, since the pH ranged from 6.8 – 7.0 in those soils. Table 7.4 % partitioning of Pb and Mn in soils with and without plant growth.  % Pb fractions in soil  Treatment /Conditions  Soil alone  Soil + Lolium  Soil + Festuca  Soil + Poa  % Mn fractions in soil  Exch  Oxide  Organic  Residual  Exch.  Oxide  Organic  Residual  B0  2.2  35  22.8  40  11  43.5  11  34.5  BA  4.2  31  25.8  39  19.2  40.5  11.3  29  B0  0.31  38  17  45  7  52  7.1  34  BA  0.51  30  25  45  11  49  19.1  21  B0  0.63  36  23.37  40  9.6  54  8.5  28  BA  0.83  40  21.17  38  15  50  8.2  27  B0  0.14  29.9  24  46  11  45  11.2  33  BA  0.19  42.3  16.51  41  16  44  14  26  The major partitioning of each metal in a soil is in bold. n=3. F significant at P<0.05. B0 – Initial soil, BA – Spiked soil.  140  % Pb fractionation  100% 80% 60% 40% 20%  Amendments alone  Amendments + Lolium  Amendments + Festuca  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  0%  Amendments + Poa  % Mn fractionation  100% 80% 60% 40% 20%  Amendments alone  Amendments + Lolium  Exch  Oxide  Amendments + Festuca  Organic  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  BALPO  BAO  BAP  BAL  0%  Amendments + Poa  Residual  Figure 7.1 Mn and Pb fractionation in soils. B0 – Initial soil, BA – Spiked soil, BAL Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Distribution of Mn in different forms in soil by the combined addition of amendments (BALPO) was: Mnoxide > Mnresidual > Mnorganic > Mnexchangeable. (Figure 7.1). A similar observation of high affinity of Mn towards the oxide phase was reported by Navas and Lindhorfer (2005). Lime application (BAL) lowered the exchangeable Mn fraction by 40 % while phosphate addition (BAP) increased the exchangeable Mn fraction by 35 %. The combined application of amendments (BALPO) lowered the exchangeable Mn fraction by almost 50 % irrespective of plant species. Soluble, exchangeable and chelated forms are the mobile and bio-available 141  fractions in soils (Thangavel. and Subhuram, 2004). Application of phosphates and compost considerably reduced the exchangeable fraction of Pb, whereas for Mn, they had an increasing effect on the mobile Mn fraction. The success of immobilisation by phosphate addition is based on chemical immobilization of the metal contaminant through the formation of insoluble heavymetal phosphate compounds in the soils, such as pyromorphite for Pb. The immobilisation mechanism is considered to be the dissolution of the Pb compounds followed by the precipitation of Pb phosphate. Thus, successful immobilisation of Pb in soil requires enhancing the solubility of soil Pb and P by decreasing soil pH and applying sufficient phosphorus so as to provide free phosphate ions (H2PO41– and HPO42-) in the soil solution (Ma et al., 1995). By the addition of phosphates in the present study, there was an increase of 35 - 40 % available P in the soil which is obviously the soluble P fraction facilitating the formation of lead phosphates and thereby Pb immobilisation. Addition of compost (BAO) may lead to complexation of Pb with soil organic matter (humic and fulvic acids), which is then adsorbed onto soil solids. Humic and fulvic acids present in compost are known to enhance the metal adsorption capacity of mineral surfaces through the formation of ternary mineral surface-metal-organic ligand complexes (Arias et al., 2002). These mechanisms may facilitate attachment of substantial amounts of Pb, accounting for the low mobility of lead in soils (Harrison and Laxen, 1981). Even though organic matter is an important reactive component in soils capable of retaining the metal cations and a good sorbent for metals (Ge and Hendershot, 2005), the degree of immobilisation depends on the level of humification of the organic matter. Similar observations of metal immobilisation by increased metal sorption in mineral-humic mixtures were reported in soils with podzolic characteristics due to the acidic soil environment and high organic matter status (Ge and Hendershot, 2005). Since the aim of the present study was to explore phytoremediation based on mobility/bioavailability of soil metals, the total metal concentrations of soil after amendment treatment and plant growth are less significant, and the observed decrease often within the range of experimental error. Phytostabilisation is not a strategy for removal of metal-contaminants, but it results in in situ inactivation or immobilisation of contaminants. The results from this study show that the exchangeable or mobile fraction of Pb and Mn decrease significantly due to growth of Poa and Lolium in conjunction with combined amendments.  142  7.3.4 Relationships between soil pH and soil metal fractions.  350  Mn fractions (mg/kg)  Pb fractions (mg/kg)  120 100  (a)  80 60 40 20 0  300  (b)  250 200 150 100 50 0  5  5.5  6  6.5  7  7.5  8  5  5.5  6  6.5  Soil pH  7  7.5  8  Soil pH Exch.  Oxide  Organic  Residual  Correlations between soil pH and soil metal fractions Exchangeable  Oxide  Organic  Residual  Pb  -0.045  0.363  0.068  0.168  Mn  -0.658*  0.707*  0.113  0.125  * R statistically significant at P < 0.05. Figure 7.2. Relationship between soil pH and metal fractions in soil, (a) soil pH and Pb fractions in the soil (b) soil pH and Mn fractions in the soil. Correlation studies conducted between different metal fractions and soil pH (Figure 7.2) have shown that the exchangeable fractions of both Pb and Mn are negatively correlated with the pH, whereas other fractions (oxide, organic and residual) are positively correlated. Exchangeable Mn gave a significant negative correlation with soil pH (R = -0.658). A significant positive correlation between pH and oxide fraction was observed for Mn (R = 0.707) whereas for Pb, correlation obtained was not significant (R = 0.363). No significant correlations between pH and organic and residual fractions were observed for either Mn or Pb, indicating that the organic and residual fractions were not significantly affected by soil pH.  143  7.3.5 Relationships between soil metal concentrations and the Enrichment Coefficient (EC)  Correlation studies conducted between soil metal concentrations and the Enrichment Coefficient (EC) of plants (Table 7.5) revealed that there was a negative correlation between total soil Pb and ECroot and ECshoot for Pb (R = -0.496 and -0.869). In the case of Mn, a moderate and positive correlation was obtained between total soil Mn and ECroot (R = 0.308), while with ECshoot a low correlation was obtained (R = 0.254. This clearly reveals the fact that total metal concentration in the soil cannot be used as a measure to judge the impact of metal contamination on the environment. High metal concentrations in the soil do not always indicate correspondingly high levels of these metals in the plants, since this depends on several factors, such as pH, cation exchange capacity, organic matter, humidity and others (Albasel and Cottenie, 1985). Table 7.5 Relationship between Enrichment Coefficient (EC) and total soil metal concentration (mg/kg). Metal Pb Mn  Relationship  Regression equation  ECR & total soil Pb ECS & total soil Pb ECR & total soil Mn ECS & total soil Mn  y= -0.269Ln(x) + 1.637 y = -0.173Ln(x) + 0.948 y = 2.468Ln(x) - 11.339 y = 0.9685Ln(x) - 3.072  Correlation Coefficient (R) -0.496 -0.869* 0.308 0.254  ECR – Enrichment Coefficient (Root), ECS – Enrichment Coefficient (Shoot) *correlation coefficient significant at P<0.05.  144  7.3.6  Relationships between soil metal fractions and plant metal concentrations  80  Root Pb (mg/kg)  Root Pb (mg/kg)  80 60  (a) 40 20  Y = 7.36Ln(x) + 48.64 R = 0.682  40 20  0 0.0  0.5  1.0  Y = 16.69Ln(x) – 18.39 R = 0.397  0  1.5  10  Exchangeable Pb (mg/kg)  50  70  2200  1800  Shoot Mn (mg/kg)  (c)  1400 1000  Y = 232.1Ln(x) + 603.4 R = 0.589  600 200 0  10  20  30  40  (d)  1800 1400 1000  Y = 195.4Ln(x) + 486.5 R = 0.669  600 200  50  0  Exchangeable Mn (mg/kg)  10  20  30  40  50  Exchangeable Mn (mg/kg)  2200  2200  Shoot Mn (mg/kg)  Root Mn (mg/kg)  30  Oxide Pb (mg/kg)  2200  Root Mn (mg/kg)  (b)  60  (e)  1800 1400 1000  Y = 702.8Ln(x) – 2754.2 R = 0.423  600 200  Y = 829.03LN(x) – 3530 R = 0.596  1800  (f)  1400 1000 600 200  100  150  200  250  300  350  100  Oxide Mn (mg/kg)  150  200  250  300  350  Oxide Mn (mg/kg) B0  BA  BAL  BAP  BAO  BALPO  Figure 7.3 Relationship between plant metals and soil metal fractions. (a) Exchangeable Pb and Root Pb, (b) Oxide Pb and Root Pb, (c) Exchangeable Mn and Root Mn, (d) Exchangeable Mn and Shoot Mn, (e) Oxide Mn and Root Mn, (f) Oxide Mn and Shoot Mn Correlation studies conducted between soil metal fractions and plant metals (Figure 7.3) revealed that in the case of Pb, there was a positive and significant correlation (P<0.05) between Pb root and Pb exchangeable in the soil (R = 0.682) and a moderate correlation between Pbroot and Pb oxide (R = 0.397). In the case of Mn, there was a significant positive correlation (P<0.05) between Mn exchangeable and Mnplant (both root and shoot) (R values, being 0.589 and 145  0.669 respectively). With Mnoxide also, Mnplant (both root and shoot) gave positive and high correlations (R values, being 0.423 and 0.596 respectively). This again confirms the mobility and bioavailability of the exchangeable and oxide fractions of Pb and Mn in the soil. The oxide bound metals are also available for plant uptake (Arias et al., 2002), which may be the result of the low soil pH in the present study. The quantity of metals absorbed by the plant depends on the concentration and speciation of the metal in the soil solution, together with its successive movement from the soil to the root surface and from the root to the aerial part (Patra et al., 2004).  7.3.7 Relationships between soil properties and metal uptake by plants  The influence of soil properties on Pb and Mn uptake (Figure 7.4) by the plants showed that soil pH had a negative effect (P<0.05) on both Pb and Mn uptake (R values being 0.569 and 0.639 respectively). Organic matter status of the soil had a positive effect on both Pb and Mn uptake (R values being 0.365 and 0.511 respectively), whereas available P status of the soil had a positive effect on Mn uptake (R = 0.405) and a negative effect on Pb uptake (R = 0.603). The negative effect of available soil P in reducing soluble Pb in the soil and decreasing Pb concentration in plants has been discussed earlier in the section “Metal concentrations and uptake in plants”. Thus the results obtained clearly reveal that the uptake of metals by plants varies greatly as a function of soil conditions (Patra et al., 2004).  The ability of Pb to bind with organic matter at low soil pH has been reported by Brown et al. (2000) in a study on Pb leaching in peat amended soil. Addition of compost along with lime and phosphate in the present study decreased the uptake of Pb and Mn, which may be the result of more sorption, complexation and precipitation of the metals as a result of the reduction in ion competition with protons in soil at near neutral pH (Yin et al., 1997).  146  Figure 7.4. Relationship between soil properties and metal uptake by the plants. (a)Pb uptake and soil pH, (b) Mn uptake and soil pH, (c) Pb uptake and % organic matter in the soil, (d) Mn uptake and % organic matter in the soil, (e) Pb uptake and available P in the soil, (f) Mn uptake and available P in the soil.  147  55  Mn uptake (micro grams/pot)  Pb uptake (micro grams/pot)  65  (a)  45 35 25 15 5 5.0  5.5  6.0  6.5  7.0  7.5  4000  (b)  3000 2000 1000  8.0  0 5.0  5.5  6.0  Mn uptake (micro grams/pot)  Pb uptake (micro grams/pot)  55  (c)  45 35 25 15 5 1.5  2.0  2.5  7.0  7.5  Soil pH  Soil pH  65  6.5  4000  (d)  3000 2000 1000 0 1.5  2.0  2.5  3.0  % organic matter in the soil  3.0  Mn uptake (micro grams/pot)  Pb uptake (micro grams/pot)  % organic matter in the soil  65  (e)  55 45 35 25 15 5 5.0  15.0  25.0  4000  (f)  3000 2000 1000 0 5.0  35.0  BA  25.0  35.0  Available P in the soil (mg/kg)  Available P in the soil (mg/kg)  B0  15.0  BAL  BAP  BAO  Relationship  Regression Equation  Soil pH vs Pb uptake Soil pH vs Mn uptake % soil organic matter vs Pb uptake % soil organic matter vs Mn uptake Available soil P vs Pb uptake Available soil P vs Mn uptake  y = -49.162Ln(x) + 124.66 y = -4485.2Ln(x) + 9798.7 y = 28.686Ln(x) + 14.812 y = 2719.4Ln(x) – 406.64 y = -14.384Ln(x) + 74.57 y = 1019.3Ln(x) – 1215.4  BALPO  Correlation Coefficient (R) -0.569* -0.639* 0.365 0.511 -0.603* 0.405  * R was statistically significant at P < 0.05.  148  Interaction of organic matter with metals is dependent on metal species and the soil pH. The protonation of negatively charged organic matter and other exchange sites at low pH will make it difficult to coordinate the metals present in soil solution (Ross, 1994).  7.4 Conclusions  •  Lowest plant concentrations and uptake values were recorded by Poa for Pb and Lolium for Mn.  •  Lime application lowered plant Pb and Mn concentrations whereas phosphate application retarded plant Pb and augmented plant Mn.  •  Addition of lime re-distributed >40% of total Pb to the oxide fraction whereas addition of phosphate re-distributed >62% of total Pb to the residual fraction.  •  Phosphate addition increased the exchangeable Mn fraction by 35% and the combined application of amendments lowered the exchangeable Mn fraction by almost 50%.  •  Compost application increased the Mn concentration (both root and shoot) in Lolium and Festuca, whereas a decreasing effect was noticed for Pb.  •  Combined amendment addition resulted in a significant decrease in Pbexchangeable (mobile) in soils growing Poa and Mnexchangeable in soils growing Lolium.  •  The re-distribution of the exchangeable fraction was mainly to the oxide fraction for Mn and residual fraction for Pb.  •  ECroot and ECshoot for Pb in Poa decreased by 72 and 60% with the combined application of amendments, while the corresponding decreases for Mn in Lolium were 48 and 43%.  •  Correlation studies conducted between soil properties and plant metal uptake revealed a positive effect of soil organic matter and available P on Mn uptake and a negative effect on Pb uptake.  This study enabled identification of the best plant-amendment combination that can reduce the mobility and phyto-availability of Pb and Mn in a highway soil, contaminated with Cu, Pb, Mn 149  and Zn due to vehicular traffic. It revealed a cost-effective package for phytostabilisation of Pb and Mn that can not only reduce hazards associated with excess Pb and Mn, but also improve soil quality and restore its functionality. Thus the environmental hazards of metal pollution depend on the metal–contaminant chemistry influenced by various geochemical and biochemical properties of soil. Application of soil amendments assist natural metal remediation of soil by hastening soil sorption processes and this effect is complemented by growing suitable plant species releasing root exudates with sequestration agents. The refinement of the technique requires molecular studies, which is suggested as the future line of work.  150  7.5 References  Albasel N. and Cottenie, A. (1985) Heavy metal contamination near major highways, industrial and urban areas in Belgian grassland. Water Air Soil Pollut., 24, 103–110. Arias, M., Barral, M. T. and Mejuto, J. C. (2002). Enhancement of copper and cadmium adsorption on kaolin by the presence of humic acids. Chemosphere, 48, 1081–1088. Arienzo, M., Adamo, P. and Cozzolino, V. (2003). The potential of Lolium perenne for revegetation of contaminated soil from a metallurgical site. Science of The Total Environment, 319, 13–25. Baker, A. J. M., McGrath, S. P., Sidoli, C. M. D. and Reeves, R. D. (1994). The possibility of in situ heavy metal decontamination of polluted soils using crops of metal-accumulating plants. Resour. Conserv. Recy., 11, 41–49. Brown, P. A., Gill, S. A. and Allen, S. J. (2000). Metal removal from wastewater using peat. Review article. Water Resour., 34, 3907–3916. Environment Canada. (1987). National inventory of sources and emissions of manganese— 1984; EPS5/MM/1, Conservation and Protection Environmental Analysis Branch, Ottawa Friedland, A. J. (1990). In Heavy Metal Tolerance in Plants: Evolutionary Aspects, Shaw, A. J., Ed.; CRC Press: Boca Raton, FL, 1990, pp. 7-19. Ge, Y. and. Hendershot, W. (2005). Modeling sorption of Cd, Hg and Pb in soils by the NICA [non-ideal competitive adsorption]—Donnan model. Soil and Sediment Contamination, 14, 53–69. Jarvis, M. D. and Leung, D. W. M. (2002). Chelated lead transport in Pinus radiata: an ultrastructural study. Environ. Exp. Bot., 48, 21-32. Joint FAO/WHO Expert Committee on Food Additives, (2000). Safety Evaluation of Certain Food Additives and Contaminants. WHO Food Additives Series 44, World Health Organization, Geneva. Harrison, R. M and Laxen, D. P. H. (1981). Lead Pollution: Causes and Control, Chapman Hall, London. Haeussling, M. and Marschner, H. (1989). Organic and inorganic soil phosphates and acid phosphatase ativity in the rhizosphere of 80-year-old Norway spruce [Picea abies (L.) Karst trees.] trees. Biology and Fertility of Soils, 8, 128-133. Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavymetals from soils. Environ. Sci. Technol., 29(5), 1232-1 238.  151  Kumpiene, J., Lagerkvist, A. and Maurice, C. (2007). Stabilization of Pb- and Cu-contaminated soil using coal fly ash and peat. Environmental Pollution, 145, 365-373. Laidlaw, M. A. S., Mielke, H. W., Filippelli, G. M., Johnson, D. L. and. Gonzalez, C. R. (2005). Seasonality and children's blood lead levels: developing a predictive model using climatic variables and blood lead data from Indianapolis, Indiana, Syracuse, New York and New Orleans, Louisiana (USA). Environ. Health Perspect., 113 (6), 793–800. Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58, 1-14. Ma, Q. Y., Logan, T. J. and Traina, S. J. (1995). Lead immobilization from aqueous solutions and contaminated soils using phosphate rocks. Environ. Sci. Technol., 29, 1118–1126. Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W. and Edwards, R. (2000). In situ metal immobilisation and phytostabilization of contaminated soils. In: T. Norman and G. Banuelos, Editors, Phytoremediation of Contaminated Soil and Water, Lewis Publishers, Boca Raton, FL pp. 323–358. Ministry of Environment, Lands and Parks. (1995). Criteria for Managing Contaminated Sites in British Columbia. Victoria, British Columbia, Queen’s Printer for B.C. Navas, A. and Lindhorfer, H. (2005) 'Chemical Partitioning of Fe, Mn, Zn and Cr in Mountain Soils of the Iberian and Pyrenean Ranges (NESpain). Soil and Sediment Contamination, 14(3), 249 – 259. Needleman, H. L., Schell, A., Bellinger, D., Leviton, A and Allred, E. N. (1990). The long-term effects of exposure to low doses of lead in childhood. An 11-year-follow-up report. N. Engl. J. Med., 322, 83-88. Padmavathiamma, P. K. and Li, L. Y. (2007). Phytoremediation Technology: Hyperaccumulation metals in plants. Water, Air, and Soil Pollution, 184, 105-126. Padmavathiamma, P. K. and Li, L. Y. (2009). Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation, 11(6), 575-590. Patra, M., Bhowmik, N., Bandopadhyay, B. and Sharma, A. (2004). Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance. Environ. Exp. Bot., 52, 199-223. Preciado, H. F. and Li, L. Y. (2006). Evaluation of metal loading and bioavailability in air, water and soil along two highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108. Raskin, I., Smith, R. D. and Salt, D. E. (1997). Phytoremediation of metals: using plants to remove pollutants from the environment. Curr. Opin. Biotechnol., 8, 221–226. 152  Ross, S. M. (1994). Retention, transformation and mobility of toxic metals in soils. In: S.M. Ross, Editor, Toxic metals in soil-plant systems, John Wiley and Sons, Chichester. pp. 63– 152. Sezgin, N., Ozcan, H. K., Demir, G., Nemlioglu,S. and Bayat, C. (2003). Determination of HeavyMetal Concentrations in Street Dusts in IstanbulE-5 Highway Environment International, 29, 979-985. Simon, L. (2005). Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300. Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612. Susarla, S., Medina, V. F. and McCutcheon, S. C. (2002). Phytoremediation: an ecological solution to organic chemical contamination. Ecol. Eng., 18, 647–658. Tessier, A., Cambell, P.G.C. and Bisson, M.(1979). Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chim., 51, 844–851. Thangavel, P. and Subhuram, C. V. (2004). Phytoextraction - Role of hyper accumulators in metal contaminated soils. Proc. Indian Natn. Sci. Acad., B, 70 (1), 109-130. US Environmental Protection Agency. Health effects support document for manganese. (2003). Available from: www.epa.gov/safewater/ccl/pdf/manganese.pdf. Accessed 1/10/04. Yin, Y., Allen, H. E., Huang, C. P. and Sanders, P. F. (1997). Interaction of Hg(II) with soilderived humic substances. Analytica Chimica Acta, 341, 73. Waldron, H. A.(1980). Metals in the environment, Academic Press, London (NY). Weis, J. S. and Weis, P. (2004). Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration. Environ. Int., 30, 685-700.  153  8.  7  RHIZOSPHERE  INFLUENCE  AND  SEASONAL  IMPACT  ON  PHYTOSTABILISATION OF METALS – A FIELD STUDY 8.1 Introduction  A wide range of pollutants such as particulates, trace elements and petroleum hydrocarbons, which originate from transportation activities, accumulate on highway surfaces in addition to direct aerial deposition. Due to the impermeability of the pavement, these pollutants are delivered by highway runoff during wet weather to roadside soils and potentially into streams via drainage networks (Thomson et al., 1997) and enter the food chain (Ma and Jennings, 2008). Of all the contaminants in the highway system, metals are the most prevalent contaminants of concern (Hathhorn and Yonge, 1996), since they are adsorbed on the soil exchange complex. High mobile concentrations lead to ecological and human health risks, as metals leach into receiving waters and enter the food chain (Alloway, 1990; Kabata-Pendias and Pendias, 2001). Highway runoff contains high concentrations of lead, zinc, copper and manganese resulting from wear of brakes, tires, and other vehicle parts and due to the introduction of methyl cyclopentadienyl manganese tricarbonyl (MMT) as a replacement for tetra-ethyl lead in 1974 (FHWA, 1998; Hall et al., 1998). While leaded gasoline has been suspended in North America and most of industrialized countries for more than three decades, residual Pb remains a concern (Hodes et al., 2003). Land associated with 12,000 km of roads in B.C. (and millions of kilometres around the world) endanger wildlife habitats where metal contamination needs to be remediated in a comprehensive manner (Precciado and Li, 2006). Environmental management of metal contamination is a challenging geotechnical and ecological problem. Currently 45 of the 50 US states and 8 of the 13 Canadian provinces and territories provide regulatory guidance on the maximum metal concentrations in surface soils, which do not yield unacceptable human health risk (Jennings and Petersen, 2006).  7  A version of this chapter will be submitted for publication. Padmavathiamma, P.K. and Li, L.Y. (2009). Rhizosphere influence and seasonal impact on phytostabilisation of metals – a field study 154  Previous studies have dealt with the identification of plant species for phytoremediation and suitabilities of different plant amendment combinations on the immobilisation of metals in soil (Padmavathiamma and Li, 2009a, b). However, verification of results under field situations is necessary. The mobile-immobile distributions of metal fractions, controlled by various pedogenic and biogenic processes, are influenced by seasonal changes (Kim and Fergusson, 1994, Duman et al., 2006) and metal-rhizosphere interactions (Jacynthe, 2007). Hence comprehensive studies are conducted under field conditions, taking into account seasonal and rhizosphere influences on soil-plant-metal contaminant chemistry, to develop Best Management Practices (BMP) for phytostabilisation of metals. The main goal of this study was field verification of the results obtained from previous pot experiments.  The study was undertaken in a Luvic Gleysolic soil, Deltaport Way along HW 17 NB ramp in Delta, BC using soil amendments of lime and phosphate in combination with three previously identified plant species, Lolium perenne, Festuca rubra and Poa pratensis (Padmavathiamma and Li, 2007). Four metals, Cu, Pb, Mn and Zn were studied and treatment efficiencies were assessed during three seasons - summer, autumn and winter. The research tasks included (1) quantifying the seasonal extent of metal accumulation in soil and assessing the seasonal impact on metal speciation in the soil as influenced by amendments and different plant species; (2) determining metal accumulation in different plant parts seasonally; and (3) assessing the influence of root-soil interactions on metal dynamics. The final outcome of the study is the development of a remediation strategy for four metals (Cu, Pb, Mn and Zn) involving suitable plants and amendments, taking into account of seasonal and rhizosphere influences, while maintaining the functional and biological integrity of soil after remediation. 8.2. Materials and methods 8.2.1 Experiment details The study area was located on the northern ramp of HW 17, Deltaport Way, Delta, B.C.  Experimental details are given in Table 1. The study was conducted as a Completely Randomized Factorial Experiment in Split plot design (Appendix D, Figure 1) with three replications. The three plant species tested individually and in combination were Lolium perenne, Poa pratensis and Festuca rubra. The amendments were lime and phosphate, selected 155  on the basis of the effect of lime on soil pH which controls metal solubility/mobility and the effect of P as a metal retardant in soils (Padmavathiamma and Li, 2008). 24 stratified randomly selected plots (1 m2) were laid out along a 100 m transect at distances of 5, 9, 11, 14 and 17 m from the edge of the ditch. The surface vegetation was removed, the soil was loosened to a depth of 15 cm and amendments incorporated with the soil. Table 8.1 Experimental program for field study Heavy metals Conditions/Treatments studied  Cu  Soil alone (T0)  Plant species  Loium perenne  Stages of sampling  90 DAS (days after sowing)   Pb   Soil plus amendments  Poa pratensis (lime and phosphate)  180 DAS (days (T1).  Mn  Festuca rubra after sowing) Lime (10 tons/ha), Phosphate (135 kg  Zn  Combination (L.  270 DAS (days P2O5/ha). Sources of lime perenne + P. after sowing). and phosphate are dolomite pratensis+F. rubra) (finely ground) and Ca HPO4. 2H2O. Design –Completely Randomized Factorial Experiment in Split Plot Design. 8 treatment combinations and three replications. Seeds sown in May 2007 and the soil and plant samples collected during three different seasons, i.e. 90 DAS, August 07 (summer), 180 DAS, November 07 (autumn) and 270 DAS, February 2008 (winter). 8.2.2 Collection of samples and laboratory analysis  The field soil was analysed for its basic physico-chemical characteristics, including pH, electrical conductivity, CEC, particle size distribution, % carbon, % nitrogen and total metal concentrations. After planting, soil samples collected during different seasons (summer, autumn and winter) were analysed for pH, electrical conductivity and total metal concentrations. During winter both rhizosphere soil (RS) and bulk soil (BS) were collected. Rhizosphere soil (RS) was removed from the roots with gentle shaking, whereas bulk soil (BS) was the soil outside the planted area in the plot where no roots are found (Gobran, et al., 2001). The metal fractionation in the soil for different seasons was estimated by the SSE procedure proposed by Tessier et al. (1979). For the plant samples, shoots and roots were separated, processed, ashed and analysed for their metal content (Padmavathiamma and Li, 2009a). Soil as well as plant extracts were analyzed for Cu, Pb, Mn and Zn using a Varian Spectre AA 220 Multi-element Fast Sequential 156  Atomic Absorption Spectrometer. Quality checks and calibrations were performed using blanks, duplicate samples and reference materials.  8.2.3 Statistical analysis  Statistical analysis was conducted using SAS version 9.1 (SAS Institute, 2001). The statistical significance of differences among means was determined by Analysis of variance (ANOVA) to compare the treatment effects on soil metal speciation, total soil metal concentration, as well as metal uptake by plants. To assess the accumulation characteristics and translocation properties of metals in plants, translocation factor (TF) and enrichment coefficient (EC) were determined. EC of root (Croots/Csoil, the ratio of root to soil concentration) and shoot (Cshoots/Csoil, ratio of shoot to soil concentration) and TF (Cshoots/Croots, ratio of shoot to root concentration) were calculated (Mattina et al., 2003; Kumar et al., 1995).  8.3. Results and discussion  The basic characteristics of the soil are given in (Table 8.2). The texture of the soil is silty clay loam and the taxonomic name of the soil according to the Canadian System of Soil Classification is Humic Luvic Gleysol. Table 8.2 Key soil characteristics before the field experiment Parameters  Values  Soil pH  5.64  Electrical Conductivity (dS/m)  0.75  % organic matter  4.5  Total nitrogen (%)  0.30  Available phosphorus (mg/kg)  6.1  Cation exchange capacity (molc kg-1)  22  Total metal concentrations (mg/kg)  Cu – 65, Pb – 98, Mn – 210, Zn - 175  Each value represents mean of three measurements.  157  8.3.1 pH and Electrical Conductivity  pH and electrical conductivity (dS/m) in soils growing different plant species, with and without treatments during three seasons are given in Figure 8.1. The lower pH values, during summer may be due to oxidation of metal salts as a result of increased aeration of the soils and removal of nutrient base cations. Release of CO2 from root respiration may reduce pH of rhizosphere soil. However, there was an increase of soil pH at 270 DAS, ie. during winter. The soil pH, initially 5.6, increased to 6.0 in Lolium growing soils, 5.8 in Festuca growing soils, 5.9 in Poa growing soils and 6.2 in soils growing a combination of these plants (1/3 each), at 270 DAS, i.e. winter. The increase in soil pH during winter may be attributed to the dilution of soil solution by higher precipitation (see Appendix D, Table 1) during the winter. When a soil solution is diluted, the concentration of H+ ions becomes diluted, causing higher pH (Jackson, 1958). Furthermore, pH changes by plants may be due to the imbalance in cation or anion release, caused by excess anion or cation uptake (Marschner, 1995). In plots where lime and phosphate amendments were applied (T1), an increase in soil pH was noticed compared to T0 (without amendments), Figure 8.1.  158  6.4  6.4  6.2  5.8 5.6  5.8 5.6  5.4  5.4  5.2  5.2  5.0  5.0  Autumn  Winter  1.0 0.9  (T0)  0.8 0.7 0.6 0.5 0.4  Summer  Autumn  Lolium  Summer  Electrical Conductivity (dS/m)  Summer  Electrical Conductivity (dS/m)  (T1)  6.0  Soil pH  Soil pH  6.2  (T0)  6.0  Winter  1.0 0.9  (T1)  0.8 0.7 0.6 0.5 0.4  Summer  Winter  Festuca  Autumn  Poa  Autumn  Winter  Combination  Figure 8.1 Seasonal influences on pH and electrical conductivity of soil. T0 – without amendments, T1 – with amendments (lime plus phosphate). Soil pH is an important parameter affecting the solubility and mobility of metal fractions, and hence ecological and human-health risks (Sreevastava and Guptha, 1996). Soil pH, sometimes called the “master variable”, has the potential to modify metal solubility/availability in several ways (McBride, 1994), including dissolution/precipitation reactions, regulating the ionisation of pH dependent exchange sites on organic matter and oxide clay minerals and influencing metal speciation in soils (Adriano et al., 2004; Sparks, 2003; Conesa et al., 2006). Chemical forms of metals in soil depend on both the source of contamination and the physicochemical properties of the soil such as pH, Eh, %organic matter and %clay. The electrical conductivity generally increased in summer, both in T0 and T1 (Figure 8.1), possibly due to the result of lower precipitation and higher evapotranspiration during this season.  8.3.2 Metal concentrations in soil  The average total metal concentrations in the soil before plant growth were 65 mg/kg for Cu, 98 mg/kg for Pb, 210 mg/kg for Mn and 175 mg/kg for Zn. Summer had an augmenting effect on 159  total metal concentrations, whereas autumn and winter had a retarding effect. However, total metal concentrations cannot be used to predict the impact of metals on the environment since only the mobile and soluble metal fraction has the potential to leach or to be taken up by plants and enter the food chain (Simon, 2005, Kumpiene et al., 2007; Mench et al., 2000). Since the bioavailability and mobility of metals depend on their association with various soil components, it is essential to quantify the metal bound to each fraction in the soil (Chopin et al., 2008).  8.3.3 Metal fractionation in soil  The fractionation of metals (%) in the soil as influenced by growth of plants, application of amendments and influence of seasons are discussed separately. The results are given in Figure 8.2. The metal fractionation in soils before plant growth is given in Table 8.3. Table 8.3. % Metal fractionation in the soil before plant growth  Metal Cu Pb Mn Zn  Exch. 10 4 12 12  % Metal fractionation Carbonate Oxide Organic 5 19 32 2 10 42 1 42 6 5 34 16  Residual 34 42 39 33  Mean values. n = 3. Before plant growth, Cu and Pb were mainly partitioned into organic and residual fractions, whereas Mn and Zn were in the exchangeable and oxide fractions. High organic matter content of the soil (4.5%) and the affinity of Cu and Pb to complex with organic ligands (Sanders et al., 1986; Ross, 1994) explain the large partitioning of Cu and Pb in the organic form. The presence of fresh organic matter can increase metal solubility, due to the availability of soluble organic compounds which form complexes with the metals (Almas et al., 1999; Shuman, 1999), whereas the humic substances in soil organic matter can reduce metal solubility by forming stable metal chelates ( Ross, 1994). Hence the extent of humification of soil organic matter governs the mobility or immobility of organic metal chelates. Carbonate fraction is unlikely in soils with pH 5.6. Extractants used in SSE are not 100% specific and there are chances of extracting non-targeted metal fractions. 160  The influence of plant growth on soil metal fractionation is given in Figure.8.2. 94% of the total Pb was found to exist in the organic, oxide and residual fractions and <5 % in the exchangeable fraction. The exchangeable fraction was significantly lowered (P <0.05) in Festuca growing soils for Cu, Lolium growing soils for Mn, and the combination (Lolium + Poa + Festuca) for Pb and Zn. This explains the suitability of the above plant species to reduce the mobile fractions of each metal studied. These results are in agreement with the previous studies with pot experiments (Padmavathiamma and Li, 2009a, b), except that the plant combination was found to be superior for reducing the exchangeable Pb and Zn under field conditions, in contrast to Poa in the pot experiments. A significant increase of the oxide fraction (P <0.05) in soils growing Festuca was observed in the case of Cu and Mn (Figure 8.3). However, in general, the oxide fraction dominated in Festuca growing soils and organic fractions in soils growing Poa and Lolium during all seasons. This may be explained by the well-developed fibrous root system of Poa and Lolium contributing to increased organic matter of soil (Padmavathiamma and Li, 2008). The differences between plant species in re-distributing metal fractions may be their inherent mechanism of responding to the soil metals by adsorption onto the root surface, absorption and accumulation in roots or precipitation within the root rhizosphere by release of root exudates (Marschner et al., 2007). The adsorption onto the root surface may be by binding of metal cations to the carboxyl functional groups on the root cell walls of the grass species. The effect of amendment application on metal fractionation in soils with different plants is shown in Figure 8.2. Application of amendments (lime plus phosphate) decreased the metalexchangeable and increased the metalcarbonate fraction, especially for Cu and Pb. This may be due to the effect of amendments on the pH of the soil.  161  Figure .8.2 % metal fractionation in soil by the influence of plants, amendments and seasons. (a) Cu, (b) Pb, (c) Mn, (d) Zn. T0 – without amendments, T1 – with amendments. L – Lolium, FFestuca, P – Poa, C – Combination (Lolium + Festuca + Poa). FS – Fallow soil. Mean values, n = 3. F significant at P<0.05 for all the four metals. SE (Standard Error) of means given in Appendix E.  162  100%  Cu fractions (%)  80% 60%  (a)  40% 20%  Pb fractions (%)  L  F  P  C  FS  L  F  P  C  FS  L  F  P  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  100%  T0  0%  C  FS  80%  Summer  Autumn  Winter  (b)  60% 40% 20%  Mn fractions (%)  L  F  80%  P  C  FS  L  F  Summer  P  C  FS  L  F  Autumn  P  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  100%  T0  0%  C  FS  Winter  60%  (c)  40% 20%  P  C  FS  L  F  Summer  P  C  FS  L  F  Autumn  P  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T0 T1  T1  T0  T1  T0  T1  T0  T1  T0  T1  F  80%  C  FS  Winter  60%  (d) 40% 20%  L  F  P  C  FS  Summer  L  F  P  C  FS  L  Autumn  Exch.  Carbonate  Oxide  F  P  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T0 T1  T1  T0  T1  T0  T1  T0  T1  T0  T1  T0  T1  0%  T0  Zn fractions (%)  L  T0  T1  100%  T0  0%  C  FS  Winter  Organic  Residual 163  Cu is less affected by changes in pH (Berggren, 1989 and Strobel et al., 2001) compared to other metals such as Zn, Mn and Pb. There are reports (Brezonik et al., 2003) that Cu is not mobilised by soil acidification. However, the ability of Cu to form chelates with organic compounds (Strobel et al., 2001) may affect the solubility of Cu, as the organic matter content of the soil is about 4.5%. The mineralization and humification processes of these compounds with time led to the fixation of Cu in the soil, converting Cu into insoluble forms. Cu has high affinity for peat moss, humin and humic acids compared to other metals and hence is more liable to form metal organic complexes, which may become unavailable at high pH (de la Rosa et al., 2003). Thus Cu when complexed by humic substances is immobile and less affected by changes in soil pH than the other three metals investigated.  The carbonate bound fraction was higher for Cu and Pb, than for Mn and Zn (Figure 8.2). The relative partitioning of metals in oxide and residual fractions of soil was higher in T1 than in T0 plots. Re-distribution in relatively immobile fractions may be due to various sorption processes: adsorption to mineral surfaces, formation of stable complexes with organic ligands, surface precipitation and ion exchange (Mench et al., 2000; Kumpiene et al., 2007). Applications of lime and phosphate amendments influence pH, redox potential, electrical conductivity and cation exchange  capacity  which  govern  sorption/dissorption,  precipitation/dissolution  and  speciation/complexation in the soil (Kumpiene et al., 2007). The enhancement of soil pH in amended soils helps to explain the decrease in metal-exchangeable (mobile metal fractions) in soils that received amendments. Phosphate enhances the immobilization of metals in soils through various processes including direct metal adsorption by phosphate, phosphate anioninduced metal adsorption, and precipitation of metals with solution phosphate as metal phosphates (Adriano et al., 2004). Precipitation as metal phosphates has been shown to be one of the main mechanisms governing the immobilization of metals, such as Pb and Zn, in soils (McGowen et al., 2001). These relatively stable metal-phosphate compounds have extremely low solubility over a wide pH range, rendering phosphate application an attractive technology for managing metal-contaminated soils (Adriano et al., 2004).  The seasonal influence of metal partitioning is evident from Figure 8.2. With the growth of the plants, the exchangeable Cu, Pb and Zn were highest in summer, declining in autumn and 164  winter. Exchangeable Mn was highest in autumn, followed by winter and summer. The availability of Mn in soil depends on its pH and oxidation-reduction potential. Reduced forms of Mn are more bio-available (Marschner, 1988). Mn can occur in more than one valence state, and the more oxidized state precipitates by the formation of hydroxides (or hydrous oxides). Up to 6.0, the hydroxides will not precipitate and solubility increases (Brady and Weil, 1996). During summer and autumn, Cu and Pb occur mainly in the carbonate, oxide and organic fractions, whereas Mn and Zn are likely to be found in the oxide and exchangeable fractions. During winter, partitioning was mainly in the oxide and residual fractions for all four metals (Cu, Mn, Pb and Zn). The re-distribution from the organic fraction to the oxide and residual fraction during winter leads to the possible formation of soluble organic metal chelates during the summer, either absorbed by the soil biota or leached to the surrounding water ecosystem (Almas et al., 1999; Shuman, 1999). During winter almost all of the metalexchangeable and metalcarbonate partitioned into the organic, oxide and residual forms (Figure 8.2). Re-distribution to the immobile fractions during winter led to decreased bioavailabilty, thereby reducing the risk of contaminant transfer and accumulation into the food chain (Kabata-Pendias and Pendias, 2001; Lee et al., 2003). Thus the bioavailable fraction (otherwise known as the mobile or labile fraction) emerges as a relevant factor in risk assessment and environmental monitoring (Adriano et al., 2004). The mobile fractions of metals, i.e. metalexchangeable and metalcarbonate were significantly reduced in soils growing Festuca for Cu, Lolium for Mn and a combination of Lolium + Poa + Festuca for Pb and Zn. Application of amendments (lime + phosphate) decreased the metalexchangeable for all four metals. During winter, the major partitioning changed to the oxide and residual fractions for each of these metals, leading to decreased bioavailability, reducing the risks of contaminant transfer and accumulation into the food chain. As stated above, remediation methods applicable to soils contaminated with metals are based on two approaches: removal/extraction of the metals from the matrix or reduction of metal mobility or bio-availability in soil (Smith and Bradshaw, 1979; Simon, 2005). When metalexchangeable is decreased in soil, the solubility or bioavailability of metals is reduced, minimising the impact on soil organisms and plants, thereby reducing the exposure pathways (Simon, 2005; Mench et al. 2000). The bioavailability of metallic ions not only depends on the solubility, but also on the thermodynamic activity of the uncomplexed ion (Petrangeli et al. 2001).  165  8.3.4 Metal concentrations and uptake in plants  Metal concentration, expressed as accumulation per unit weight (mg/kg dry weight), and metal content, which denotes the total uptake or removal/m2 are discussed separately. Metal concentrations in different plant species, with and without application of amendments for three seasons are provided in Table 8.4, whereas the metal uptake by plants is given in Figure 8.3. The supply of ions to plants is controlled by the kinetics of solubilisation of ions adsorbed to the solid phase of soil (Chaney et al. 1997). The metal concentrations in plants (mg/kg) with and without application of soil amendments during summer, autumn and winter are given in Table 8.4. Without application of amendments (T0), the lowest metal concentrations were as follows: Lolium for Cu and Mn, and the combination for Pb and Zn. With the application of amendments (T1), Festuca recorded the lowest concentrations for Cu, Lolium for Mn and the combination for Pb and Zn (Table 8.4). The same trend was found for the metal uptake, except for Zn, for which Poa gave the lowest value in T1 plots. Irrespective of plant species, the root concentration was higher than shoot concentration for all four metals studied. This is consistent with the findings from previous pot experiments (Padmavathiamma and Li, 2008). The partitioning of metals into various soil chemical pools by plant growth (Figure.8.2) is reflected in the plant metal concentrations, in agreement with reports by Brooks (1998) and Weis and Weis (2004) that plant growth significantly influences metal speciation in soil, contributing to the mobility or phyto-availability of metals.  166  Table. 8.4. Metal concentrations in plants (mg/kg), (n = 3, mean values ± S.D). F significant at P <0.05 Summer Autumn Winter Metal Plant species Root Shoot Root Shoot Root Shoot Without application of amendments (T0) Lolium 51±10 24±4 57±16 16±5 66±4 11±3 Cu Festuca 86±15 26±8 78±17 28±4 89±10 23±7. Poa 66±21 23±12 62±12 20±7 77±14 16±5 Combination 61±9 28±8 58±18 24±10 69±11 20±7 Pb  Mn  Zn  Cu  Pb  Mn  Zn  Lolium Festuca Poa Combination  24±4 19±6 17±8 12±7  9±1 7±1 5±2 4±3  28±3 20±4 19±2 13±6  13±3 10±4 9±0.6 7±0.9  31±7. 25±3 26±9 20±3  6±4 5±2 4±1 3±0.4  Lolium Festuca Poa Combination  65±28 87±18 112±40 99±34  35±11 76±9 104±17 92±13  99±30 104±21 123±17 118±18  73±9 87±11 111±10 97±20  72±15 89±22 134±23 124±28  62±18 54±18 82±23 79±19  Lolium Festuca Poa Combination  71±6 79±13 66±11 57±14  31±8 29±13 25±4 19±13  Lolium Festuca Poa Combination  63±15 50±12 68±5 39±5 69±11 51±9 75±9 41±4 47±11 38±7 58±13 32±12 40±13 30±8 48±11 25±10 With application of amendments (T1) 47±14 21±2 51±8 18±6 40±8 18±4 47±16 16±3 54±16 23±11 58±2 20±8 56±8 26±3 51±7 24±6  58±8.8 51±13 61±6.7 59±9.9  16±2.5 13±4 17±4.9 19±4.7  Lolium Festuca Poa Combination  16±3 15±6 13±7 9±3  7.8±0.1 6.9±2 3.7±0.9 2.3±0.6  17±3 22±10 14.9±7 13.5±8  10±1.9 9±0.9 7±0.3 5±0.6  19±3 22±6 16±4 18±8  3±0.4 5.3±0.8 2.3±0.3 1.9±0.2  Lolium Festuca Poa Combination  49±20 67±11 101±23 89±12  38±14 57±12 89±20 82±5  69±13 78±15 112±13 104±15  59±13 66±10 109±18 84±9  97±19 102±16 121±15 110±19  35±6 42±16 79±21 66±13  Lolium Festuca Poa Combination  56±12 61±13 36±6 29±9  42±15 46±9 28±7 25±7  62±13 70±11 49±8 40±10  33±12 40±11 29±6 20±9  69±8 73±4 58±2 51±14  27±6 30±7 20±3 16±11 167  Metal uptake by plants during three seasons is portrayed in Figure 8.3. The shoot uptake was highest in summer for Cu and Zn, whereas it was highest in autumn for Pb and Mn. The root uptake was found to be highest during winter for all the four metals studied. The same trend, shown for metal concentration (Table 8.4) and uptake (Figure 8.3), reveals that plant biomass did not contribute to the accumulation of metals in the plants. During winter, the shoot concentration for Cu was only 1/6th of the root concentration, and for Pb it was only about 1/10th. Variations in the seasonal pattern of uptake potential of metals may be due to the growth effect and other factors such as the quantity of rainfall, abiotic factors such as temperature and variations in metal concentrations in the environment. The lower Mn uptake during summer may be due to the increase in Mn-oxidizing bacteria in the rhizosphere (Arines et al., 1992), which may reduce the oxidation-reduction potential and availability of Mn in the rhizosphere. The changes in plant metal concentrations and uptake may be due to the growth effect, changes in metal concentrations in the environment during different seasons as well as abiotic factors such as temperature, precipitation etc.  168  Figure 8.3. Metal uptake by the plants during three seasons. (a) – Cu uptake, (b) – Pb uptake, (c) – Mn uptake, (d) – Zn uptake. Mean values. n = 3, F significant at P <0.05. L- Lolium, F – Festuca, P – Poa, C- Combination (.Lolium + Festuca + Poa). S – summer, A – autumn, W – winter. T0 – without amendments, T1 – with amendments.  169  Cu uptake (micro grams/sq.m)  1200  Pb uptake (micro grams/sq.m)  500  Mn uptake (micro grams/sq.m)  9000  1000 800  (a)  600 400 200 0  T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L  450  F  400  P  C  L  F  S  P  C  L  F  A  P  C  W  350  (b)  300 250 200 150 100 50 0  T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 8000  L  F  P  C  L  F  S  7000  P  C  L  F  A  P  C  W  6000  (c)  5000 4000 3000 2000 1000 0  Zn uptake (micro grams/sq.m)  T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 2500  L  F  2000  P  C  L  F  S  P  C  L  F  A  P  C  W  1500  (d)  1000 500 0  T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L  F  P  C  L  S  F  P A  Root  C  L  F  P  C  W  Shoot 170  Even though the shoot uptake was less than the root uptake for all four metals studied, the % translocation to the above-ground portions was higher for Mn and Zn than for Cu and Pb. Lime and phosphate amendments decreased both root and shoot uptake of all four metals. These soil amendments influence key processes and factors that control the dynamics of trace elements in soil, thereby assisting natural attenuation of metal contaminants (Adriano et al., 2004). These changes in soil properties modify soil processes, resulting in mobilisation or immobilisation and regulating the entry of metals into the plants. Stable metal-phosphate compounds formed by the application of phosphate amendment, especially in the case of Pb and Zn, have extremely low solubility over a wide pH range, reducing the entry of metals into the plants in metalcontaminated soils (Adriano et al., 2004; Simon, 2005). The uptake of Cu, Mn, Pb and Zn by plants is often found to decrease with liming, which is attributed to the increased adsorption/precipitation at high pH (Geebelen et al., 2002; Cox and Rains, 1972). There are differing reports with respect to metal accumulation in plants during different seasons. Some scientists (Kim and Fergusson, 1994; Brekken and Steinnes, 2004) have stated highest metal contents (Cd, Cu, Ni, Pb, Sn, Zn) during autumn and relatively low levels during the spring, whereas others (Wilkins, 1978; Martin and Coughtrey, 1982) have indicated the highest foliar levels during spring and the lowest during winter. Cacador et al. (2000) observed more heavy metal accumulation in Spartina maritima and Halimione portulacoides in summer. Djingova and Kuleff (1994) established that the developmental stage was the most significant explanatory factor for heavy metal accumulation in shoots, since it may be the stage of maximum biomass production.  8.3.5 Metal accumulation charecteristics in plants  The metal accumulation pattern in plants as explained by EC (Enrichment Coefficient) for roots and shoots and TF (Translocation factor) are given in Table 8.5. The seasonal influence on the accumulation pattern is given in Figure 8.4.  171  Table 8.5. Enrichment Coefficient (ECroot and ECshoot) and Translocation Factor (TF) of metals with and without amendments  Metal  Cu  Plant species  ECS  TF  ECR  ECS  TF  Lolium  0.74  0.36  0.41  0.55  0.26  0.35  Poa  0.54  0.29  0.45  0.84  0.28  0.34  Festuca  1.12  0.38  0.44  0.49  0.21  0.31  Combination  0.66  0.32  0.43  0.54  0.23  0.42  *  NS  NS  *  NS  *  Lolium  0.24  0.06  0.23  0.2  0.05  0.21  Poa  0.2  0.05  0.22  0.15  0.04  0.14  Festuca  0.21  0.04  0.19  0.22  0.02  0.19  Combination  0.15  0.04  0.25  0.11  0.03  0.17  *  NS  NS  *  NS  *  Lolium  1.23  1.17  0.78  1.1  1.01  0.53  Poa  2.63  1.38  0.92  1.58  1.29  0.78  Festuca  1.27  1.13  0.91  1.13  1.07  0.88  Combination  1.61  1.42  0.91  1.25  1.17  0.8  *  *  *  *  *  *  Lolium  0.68  0.42  0.77  0.45  0.38  0.71  Poa  0.51  0.29  0.70  0.40  0.25  0.63  Festuca  0.71  0.49  0.78  0.59  0.45  0.73  Combination  0.44  0.27  0.69  0.41  0.26  0.61  *  *  NS  *  *  *  F  Mn  F  Zn  F  with amendments (T1)  ECR  F  Pb  without amendments (T0)  Mean values, n = 3. * F significant at P<0.05. NS – not significant. From EC and TF values (Table 8.5), it is observed that ECshoot and TF were significantly decreased (P<0.05) by the application of lime and phosphate amendments. This suggests that lime not only increased the soil pH, favouring the sorption of metals in the soil, but also inhibited the translocation of metals, especially Pb from root to shoot (Basta and Tabatabai, 1992). The lowest ECR, ECS and TF values were recorded in Festuca for Cu, Lolium for Mn and Combination for Pb and Zn (Table 8.5). 172  Metal concentration ratio  3 Cu  2.5  Pb 2  Mn Zn  1.5 1 0.5 0  ECR  ECS Summer  TF  ECR  ECS Autumn  TF  ECR  ECS  TF  Winter  Figure 8.4. ECR (Enrichment Coefficientroot), ECS (Enrichment Coefficientshoot), TF (Translocation Factor) of metals during different seasons. Mean values, n = 3. F significant at P<0.05 for all the four metals. Application of amendments (lime plus phosphate) significantly reduced the EC and TF. ECshoot was highest during summer for Cu and Zn, but during autumn for Pb and Mn (Figure 8.4). ECroot was highest in winter for all four metals. TF of each of these metals was lowest during winter (Figure 8.4), indicating that there was minimum transport of metals to the above-ground portion, with enrichment of metals in the roots. The translocation of Cu and Pb to the above-ground portion was only 20-30% of the root accumulation, whereas for Mn and Zn, it was 60 – 80 %of the root accumulation. The ultimate objective of a remediation process must be not only to remove/immobilise the metal pollutants from the soil, but most important, to sustain soil health, i.e. the continued capacity of soil to function as a vital living system, sustaining biological productivity, promoting the quality of air and water, and maintaining plant, animal, and human health (Doran and Safley, 1997). In this context, using natural amendments and plant species to accelerate natural soil attenuation/remediation has an edge over other remediation strategies. In general, the EC and TF values should be <1 for phytostabilisation and >1 for phytoextraction (Brooks 1998). In the present study, the EC and TF of studied plants were <1 for all metals except Mn. Even if 173  phytostabilisation cannot be considered as a clean-up method, this study indicates that it can reduce the inherent risk associated with a contaminated site based on a reduction of the soil mobile and bioavailable metal fraction.  8.3.6 Bulk soil (BS) vs Rhizosphere soil (RS) 8.3.6.1 pH  Soil pH was found to be higher and electrical conductivity lower in the bulk soil (BS) compared to those in the rhizosphere soil (RS) (Figure 8.5).  6.4 Rhizosphere soil  6.2  Soil pH  Bulk soil  6 5.8 5.6 5.4  L  F  P  C  Figure 8.5 pH of Rhizosphere soil and Bulk soil. L – Lolium, F – Festuca, P- Poa, C Combination. Mean values, n = 3. F significant at P<0.05. Data set is the mean of T0 and T1. SE of mean = 0.45. The difference in soil pH between BS and RS may be due to root exudation, microbial respiration and unequal uptake of cations and anions by plant roots (Lombi et al., 2001). Since plants absorb most mineral nutrients and metals as ions, imbalances in the absorption of cations and anions result in root excretion of compensating H+, OH- and HCO3- ions into the rhizosphere causing pH changes (Lombi et al., 2001). Thus differences between the pH of rhizosphere soil and bulk soil can cause processes of adsorption or desorption and precipitation or solubilisation of metals (Lombi et al., 2001). Redox potential can also change pH in the rhizosphere as a consequence of the release of reducing agents by microbial activity. Plant roots release about  174  17% of plant photosynthates into the soil with resulting enhancement of microbial populations and activity (Patra et al., 2006).  8.3.6.2 Metal Fractionation in RS and BS  The metal fractionation in RS and BS is given in Figures 8.6. In general, exchangeable and organic fractions were higher in RS compared to in BS, whereas, oxide and residual fractions were higher in BS than in RS. The organic fraction dominates the rhizosphere soil in the case of Cu, whereas oxide fraction dominates the RS for the other three metals, ie. Pb, Mn and Zn. Residual fraction dominates the BS in the case of all the four studied metals. Even though exchangeable fraction is higher in RS than BS, in certain cases such as Festuca growing soils for Cu, Lolium growing soils for Mn and Combination growing soils for Pb and Zn, the exchangeable metal fraction was higher in BS when compared to RS. The dominance of the exchangeable fraction in BS when compared to that in RS may be attributed to the sequestering action of root exudates of the corresponding plants resulting in redistribution of mobile fractions to immobile forms. The rhizosphere is a very dynamic environment governed by the reaction between its various components: soil, plant and micro organisms. This difference in metal fractionation can be attributed to the rhizosphere effect which accounts for the increased microbial biomass and activity occurring in the immediate vicinity of roots. This phenomenon is largely due to the high flux of carbon originating from root exudation (Lombi et al., 2001).  175  Figure 8.6. Metal fractions in Rhizosphere soil and Bulk soil. (a) Cu, (b) Pb, (c) Mn, (d) Zn. RS – Rhizosphere soil, BS – Bulk soil. T0 – without amendments, T1 – with amendments. Mean values, n = 3. F significant at P<0.05. SE (Standard Error) of means given in Appendix E.  176  100%  Cu fractions (%)  80%  (a) 60% 40% 20% 0%  100%  RS  BS  RS  T0  T1  RS  BS  RS  T0  Lolium  80%  Pb fractions (%)  BS  BS  RS  T1  BS  RS  T0  BS T1  Festuca  RS  BS T0  Poa  RS  BS T1  Combination  (b)  60% 40% 20% 0%  100%  RS  BS  RS  T0  RS  T1  80%  Mn fractions (%)  BS  BS  RS  T0  BS  RS  T1  Lolium  BS  RS  T0  BS  RS  T1  Festuca  BS  RS  T0  Poa  BS T1  Combination  60%  (c) 40% 20% 0% 100%  RS  BS  RS  T0  Zn fractions (%)  80%  BS T1  RS  BS  RS  T0  Lolium  BS  RS  T1  BS  RS  T0  BS T1  Festuca  RS  BS T0  Poa  RS  BS T1  Combination  (d)  60% 40%  20%  0% RS  BS  RS  T0  BS T1  Lolium  RS  BS  RS  T0  BS  RS  T1  Carbonate  RS  T0  Festuca  Exch  BS  T1 Poa  Oxide  BS  Organic  RS  BS T0  RS  BS T1  Combination  Residual 177  Root exudates include a wide spectrum of organic substances which directly affect the chemical properties of the rhizosphere and in particular the speciation and bio-availability of metals. In addition to root exudates, plant roots themselves can alter the various soil processes which have a major influence on the metal dynamics in the soil (Marschner, 1995).  8.3.6.3. Total metal concentrations in RS and BS  Total metals (mg/kg)  160 140 120 100 80 60 40 20 0  RS  BS  RS  Cu  BS  RS  Mn Lolium  Festuca  BS Pb  Poa  RS  BS Zn  Combination  Figure 8.7. Total metal concentrations in Bulk soil and Rhizosphere soil. RS – Rhizosphere soil, BS – Bulk soil. Mean values, n = 3. F significant at P<0.05 for all the four metals. SE of means: Cu – 4.67, Mn – 14.04, Pb – 4.13, Zn – 11.8. Variations were observed between the total metal concentrations in the rhizosphere soil and bulk soil (Figure 8.7). For Cu and Zn, the concentration in RS of Festuca growing soil was less than that in BS, whereas for Mn, the concentration in RS of Poa growing soil was lower than in BS. In the case of Pb, concentration in RS was lower than that in BS for all plant species. The metal concentration in RS of a particular plant species is governed by the demand of the metal by the plant species. If the demand is high, concentration will be low in RS compared to in BS (Gobran et al., 2004). The potential ligands in the rhizosphere of each plant species are selective for particular metals, which finally controls the metal speciation and bio-availability (Jacynthe, 2007).  178  Root-soil interactions can strongly influence the soil solution chemistry in the rhizosphere (Ksouri et al., 2007). Understanding the rhizosphere influence on the solubility and mobility of metals helps in improving soil remediation techniques such as phytostabilisation. When the plants undergo stress conditions such as deficiencies of nutrients or toxicities of contaminants, biochemical pathways are initiated which cause plant roots to respond by secreting chemicals from the roots to the soil (Salisbury and Ross, 1992). Root exudates may include protons, HCO3ions, organic acids such as formic acid, acetic acid, citric acid, malic acid etc. These secretions tend to increase nutrient availability and decrease availability of toxicants (Salisbury and Ross, 1992). The ways in which plant roots alter the local chemistry in the rhizosphere controls the metal mobilisation or immobilisation in the soil (Marschner et al., 2007). This explains the differences in the characteristics of rhizosphere soil (RS) and bulk soil (BS), brought out by changes in soil pH, exudation of enzymes and low molecular weight organic molecules, bulk density and water content (Gregory and Hinsinger, 1999). Ten to forty % of the total net C assimilated by plants is released in the form of soluble root exudates and also insoluble materials such as cell walls and mucilage (Marschner, 1998). The release of root exudates provides a conducive microclimate in the rhizosphere by triggering the soil biology and increasing the metal flux into that region. Increased sorption of metals onto bacteria, fungi, organic matter, and plant roots in the rhizosphere leads to retention of metal-pollutants and reduce the exposure to the environment, thereby reducing the existing and associated risks due to metal contamination. However the sustainability of this technique requires long term experiments on phytostabilisation, is a suggested future line of work. .  179  8.4 Conclusions  The important conclusions drawn from the study are as follows –  •  An increase in soil pH and a decrease in electrical conductivity (EC) in plots were observed from summer to winter.  •  Application of soil amendments decreased the exchangeable fraction and plant uptake of all the four metals, Cu, Mn, Pb and Zn. Exchangeable Cu, Pb and Zn were highest in summer while exchangeable Mn was highest in autumn.  •  Lowest mobile fractions (exchangeable. and carbonate bound) were observed in soils growing Festuca for Cu, Lolium for Mn, and the combination (Lolium, Poa and Festuca) for Pb and Zn.  •  During summer and autumn, major partitioning was in the organic and oxide fractions for Cu and Pb, while it was in the oxide and exchangeable fractions for Mn and Zn. During winter, it was in the oxide and residual fractions for all the four metals.  •  Shoot concentrations were highest in summer for Cu and Zn and highest in autumn for Pb and Mn. Root concentrations were highest in winter for all the four metals.  •  Lowest metal concentrations were observed in Festuca for Cu, Lolium for Mn and the combination (Lolium + Poa + Festuca) for Pb and Zn, with addition of soil amendments.  •  Soil pH was higher and electrical conductivity lower in the bulk soil when compared to the rhizosphere soil.  •  Partitioning of metals was mainly in the oxide and residual fractions in the bulk soil, whereas it was in the exchangeable and organic fractions in the rhizosphere soil.  The results clearly demonstrate the effectiveness of growing Lolium perenne L, Festuca rubra L and Poa pratensis L with soil amendments (lime + phosphate) for the phytostabilisation of metal-contaminants (Cu, Pb, Mn and Zn) in the highway soils of southwest British Columbia. One critical aspect largely missing and requiring more research is the long-term success of phytostabilisation.  180  8.5 References  Adriano, D. C., Wenzel, W. 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(1999). Organic waste amendments effect on zinc fractions of two soils. J. Environ. Qual., 28, 1442–1447. Simon L. (2005): Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300. Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612. Sparks, D. L. (2003). Environmental Soil Chemistry, second edition. Academic Press, San Diego, California, p. 368. Sreevastava, P. C. and Gupta, U. C. (1996). Trace elements in crop production. Science Publishers, Inc., Lebanon, USA. Strobel, B. W., Hansen, H. C. B., Borggaard, O. K., Andersen, M. K. and Raulund-Rasmussen, K. (2001). Composition and reactivity of DOC in forest floor soil solutions in relation to tree species and soil type. Biogeochemistry, 56, 1–26. Tessier, A., Cambell, P. G. C. and Bisson, M. (1979). 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The speciation of metals and the physico-chemical properties of the soil are significant in determining bio-availability. Soil characteristics (eg. pH, clay, organic matter content and type, moisture content etc.) control the speciation of metals, temporary binding by particle surfaces (adsorption-desorption processes), precipitation reactions and availability in soil solution (Adriano et al., 2004). This research project was conducted to develop an effective and environmentally friendly technology to effectively limit the dispersal of metal contaminants to the surrounding natural environment, while maintaining the biological and functional integrity of the soil after remediation. The preliminary studies (Chapter 3) discussed field measurements of trace element accumulation (Cu, Pb, Mn and Zn) in soils and plants along the highway sites and investigated the phytoremediation potential of plants that spontaneously colonised the site. Concentrations of Cu, Pb, Mn and Zn decreased with increasing distance from the highway. Festuca rubra, which invaded and colonized the contaminated site, was found to be an ideal candidate for the subsequent phytostabilisation studies. The moss collected from the study site, Rhytidiadelphus squarrosus, was found to accumulate significant amounts of Pb. Information from this study provided insight to formulate the technical program for the subsequent studies. The next study (Chapters 4 and 5) evaluated the phytoextraction/phytostabilisation potential of five plant species, Brassica napus L (rape), Helianthus annuus L. (sunflower), Lolium perenne L (perennial rye grass), Poa pratensis L (Kentucky blue grass) and Festuca rubra L (creeping red fescue) for metals (Cu, Pb, Mn and Zn) in soils with different metal contamination levels. Soil metal (Cu, Pb, Mn and Zn) fractionation and metal accumulation characteristics in plants were studied at two stages of plant growth, 90 and 120 DAS (days after sowing). In soils with total Cu, Pb, Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg respectively, none of the seeds germinated, maybe because of metal toxicities (Adriano, 2001).  186  In the study investigating the accumulation characteristics and translocation properties of metals (Cu, Pb, Mn and Zn) in different plant species (Brassica napus, Festuca rubra, Helianthus annuus, Lolium perenne, and Poa pratensis ) (Chapter 4), Enrichment Coefficients (EC) of root and shoot and Translocation Factors (TF) were discussed in addition to metal absorption and metal uptake values. The metal concentrations in plants increased by nearly a factor of two for Cu and Pb, three times for Mn and four times for Zn in BA soils, compared to B0 soils. The efficiency of plants to accumulate metals followed the order, Festuca > Lolium > Helianthus > Poa > Brassica for Cu, Helianthus > Brassica > Festuca > Poa > Lolium for Pb, Poa > Festuca > Lolium > Brassica > Helianthus for Mn and Helianthus > Festuca > Poa > Brassica > Lolium for Zn. Metal removal was higher at 120 DAS than at 90 DAS, and metals concentrated more in below-ground tissues with less translocation to the above-ground parts. EC (Enrichment Coefficient) values indicate that Festuca had the highest accumulation for Cu, Helianthus for Pb and Zn, and Poa for Mn. Metal uptake values were lowest for Festuca and highest for Lolium among the plant species studied, demonstrating that metal content or uptake is more important than metal concentration in a phytoremediation study. The effect of plant growth on the re-distribution of metal fractions (Chapter 5) at two different stages of plant growth (90 and 120 DAS) showed that there was a decrease in the exchangeable fraction and an increase in the oxide and organic fractions of metals in soils. The oxide fraction of metals dominated in Festuca soils, organic fraction in Lolium and Poa soils and exchangeable fraction in Helianthus and Brassica soils. There was a significant partitioning of metal fractions to insoluble forms by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. The relative partitioning of metals shifted more to the immobile forms as the growth of plants advanced from 90 to 120 DAS. The percentage migration by leaching from the soil was more for Cu and Pb than for Mn and Zn. Based on the results, Lolium, Poa and Festuca were identified to be suitable for phytostabilisation of Cu, Pb, Mn and Zn in moderately contaminated acid soils. Chapter 6 outlines the effect of soil amendments in modifying soil properties and thereby influencing the plants to immobilise Cu and Zn. Plant species Lolium perenne, Festuca rubra and Poa pratensis were tested in the presence of soil amendments (lime, phosphate and compost, both individually and in combination) to assess the effect of soil-plant-amendment interaction on phytostabilisation of Cu and Zn. Changes in soil pH due to the application of lime had a significant effect on the exchangeable fractions of Cu and Zn, organic fractions of Cu and oxide 187  fractions of Zn in soil. Maximum metal immobilisation was achieved in the soil by the combined application of amendments (lime + phosphate + compost), in conjunction with growth of Festuca for Cu and Poa for Zn. Application of lime significantly reduced the exchangeable fraction of Cu and Zn. Phosphate application increased the exchangeable Cu content of soil and enhanced plant Cu, whereas it led to decreased plant Zn. Lowest EC and TF values were observed in Festuca for Cu and Poa for Zn, with the combined application of amendments. Zn exhibited the highest mobility, as shown by exchangeable Zn fraction in the soil. It is likely that its accumulation will remain in the highway environment for years to come due to persistent sources of this metal, e.g. tire rubber, motor oil, grease, etc. (Viklander, 1998; Varrica et al., 2003). In the study investigating the effect of plant growth and amendment addition on Pb and Mn immobilisation (Chapter 7), addition of soil amendments to accelerate physico-chemically driven sorption processes and growth of appropriate plant species to reduce physiologically driven uptake of Pb and Mn were investigated. Lolium perenne, Festuca rubra and Poa pratensis were tested in the presence of soil amendments (lime, phosphate and compost, both individually and in combination). Lime application lowered plant Pb and Mn concentrations, while phosphate application decreased the plant Pb and increased the plant Mn. Addition of lime re-distributed >40% of total Pb to the oxide fraction whereas addition of phosphate re-distributed >62% of total Pb to the residual fraction. Phosphate addition increased the exchangeable Mn fraction by 35% and the combined application of amendments lowered the exchangeable Mn fraction by almost 50%. Combined amendment addition resulted in a significant decrease in the exchangeable (mobile) metal fraction in soils growing Poa for Pb and in soils growing Lolium for Mn. ECroot and ECshoot for Pb in Poa decreased by 72 and 60% with the combined application of amendments, while the corresponding decreases for Mn in Lolium were 48 and 43%. Chapter 8 reports on the field experiment conducted at the study site to confirm the results from the pot experiments. The primary objectives of this study were: (1) to quantify the seasonal effect of metal accumulation in soil and to assess the seasonal impact on the metal speciation in the soil by the influence of soil amendments and different plant species; (2) to determine accumulation differences between sampling periods in plant parts and to identify the plant part accumulating significantly higher amounts of metals seasonally; and (3) to assess the influence  188  of root-soil interactions on metal dynamics. The final outcome of the study was the development of a remediation strategy for metals (Cu, Pb, Mn and Zn) involving suitable plants and amendments incorporating seasonal and rhizosphere influences and maintaining the functional and biological integrity of soil after remediation. Application of amendments decreased the exchangeable fraction and plant uptake of all four metals, Cu, Mn, Pb and Zn. Exchangeable Cu, Pb and Zn were highest in summer and exchangeable Mn, highest in autumn. Lowest mobile fractions (exchangeable. and carbonate bound) were observed in soils growing Festuca for Cu, Lolium for Mn, and a combination (Lolium, Poa and Festuca) for Pb and Zn. During summer and autumn, Cu and Pb were mainly partitioned in the organic and oxide fractions, whereas Mn and Zn were partitioned in the oxide and exchangeable fractions. During winter, major partitioning was in the oxide and residual fractions for all four metals. Lowest metal entry into plants was observed to occur as follows: Festuca for Cu, Lolium for Mn and the combination (Lolium + Poa + Festuca) for Pb and Zn. The soil pH was higher and electrical conductivity lower in bulk soil compared to rhizosphere soil. Partitioning of metals was mainly into the oxide and residual fractions in the bulk soil, whereas it was in the exchangeable and organic fractions in the rhizosphere soil.  The studies conducted on the remediation potential of plants in soils from two highway sites with similar geometric designs, but different conditions (i.e. climate, land use, daily traffic, geology, etc.) showed common patterns of metal fractionation in soil and metal accumulation characteristics in plants. Among the plants studied, Lolium perenne L, Festuca rubra L and Poa pratensis L are promising with respect to immobilising metal contaminants in soil and can be recommended for practical phytostabilisation. However this dissertation did not collect data related to the cost-effectiveness of phytostabilization to limit the dispersal of metal contaminants in highway soils.  9.2. Recommendations and future work  Lolium perenne L, Festuca rubra L and Poa pratensis L have been identified as being suitable for phytostabilisation of metal-contaminants (Cu, Pb, Mn and Zn) in highway soils. Significant partitioning of metal fractions to insoluble forms was achieved by the growth of Festuca for Cu, Poa 189  for Pb and Zn and Lolium for Mn. The speciation of metals and the physico-chemical properties of  the soil are significant in determining bio-availability and mobility of metals in soils rather than the total metal concentration in the soil. Addition of soil amendments such as lime, phosphate and compost complement the plant effect in metal immobilisation by maintaining a favourable soil pH, and increasing the metal sorption in soil. Growing these plant species along highway soils could help to reduce the metal concentration of highway runoff by filtering or trapping metal-containing particulates and reducing the amount of metal-contaminated sediments entering the biota. Since the distribution and association of metals with various soil fractions directly affect mobility and bioavailability (Robinson et al., 2005), continuous monitoring of metal partitioning during different plant growth stages is essential to prevent associated risks. The mobile-immobile distributions of metal fractions, controlled by various pedogenic and biogenic processes, are influenced by seasonal changes (Duman et al., 2006; Kim and Fergusson, 1994). Hence monitoring the seasonal influence on metal fractionation would help to forecast the environmental pollution from metal contaminants in the soil. Even though there is variability with respect to plant species in re-distributing metal fractions in soil (Robinson et al., 2009), considering the metal fractions of environmental importance, the proportion of metals bonded to oxides was higher than the proportion associated with organic and residual fractions. Reasons for this observation require further elaboration. The effects of combined metals on plant metal uptake are complex (Ebbs and Kochian, 1997). Further studies are required to elucidate the interactions (antagonism or synergism) between metals at different concentration levels. No attempt was made in the current work to assess the presence and concentration of the appropriate transport proteins or translocating chelating molecules in plants. It is known that these compounds play a very important role in the translocation of metals (Hiromura and Sakurai, 2001), and this should be investigated in the future studies. The metal speciation due to aging in the spiked soil and the change in microbial biomass and mycorrhization are also suggested as subjects for future studies.  190  Mechanisms may vary among different plant species to cope with metal exposure. Hence studies on metal detoxification in the plant species tested may yield a better understanding of molecular mechanisms for metal-tolerance, an important topic for future work. Identified plants should be investigated for their physiological aspects, especially stress physiology and exposure to sUVB (supplemental UVB) radiation under different levels of metalcontaminations. Characterisation of root exudates of different plant species to identify metal specific organic ligands may help to explain the differential responses of plants to metal-partitioning in soil. Impact of metal sequestration in roots and in the rhizosphere on soil micro and macro fauna and whether this will cause off-site concerns need further study. Characterization of soil in identifying its mineral or material oxide and residual fractions of metals may provide a better understanding of the fate of metal-contaminants in soil. One critical aspect largely missing at this point is the long-term success of phytostabilisation. Hence long-term studies are recommended to help evaluate the efficacy of phytostabilisation in remediating metal toxicity, in promoting plant succession, and in maintaining the functional integrity of soil after remediation.  191  9.3. References  Adriano, D. C. (2001). Trace Elements in Terrestrial Environments: Biogeochemistry, Bioavailability and Risks of Metals (Second edition), Springer-Verlag, New York. Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S.(2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142. Duman, F., Olcay, O. and Demirezen, D. (2006). Seasonal changes of metal acumulation and distribution in shining pondweed (Potamogeton lucens). Chemosphere, 65 (11), 21452151. Ebbs, S. D and. Kochian, L. V (1997). Toxicity of zinc and copper to Brassica species: implications for phytoremediation. J. Environ. Qual., 26, 776–781. Gobran, R. G., Wenzel, W. W. and Lombi, E. (2001). Trace Elements in the Rhizosphere. CRC Press, Washington DC, pp. 321. Hiromura, M. and Sakurai, H. (2001). Intracellular metal transport proteins, RIKEN Review No. 35, 23-25. Kim, N. D. and Fergusson, J. E. (1994). Seasonal variations in the concentrations of cadmium, copper, lead and zinc in leaves of the horse chestnut (Aesculus hippocastaneum L). Environmental Pollution, 86, 89–97. Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W and Edwards, R. (2000): In situ metal immobilization and phytostabilization of contaminated soils. In: Terry N., Bañuelos G. (eds.): Phytoremediation of Contaminated Soil and Water. Lewis Publ., Boca Raton, London, New York, Washington D.C, pp. 323–358. Robinson, B. H., Bolan, N. S., Mahimairaja, S. and Clothier, B. E. (2005). Solubility, mobility and bioaccumulation of trace elements: abiotic processes in the rhizosphere. In: Trace Elements in the Environment: Biogeochemistry, Biotechnology, and Bioremediation (Eds. MNV Prasad, KS Sajwan, R Naidu). CRC press, Boca Raton Florida, pp. 97 – 110. Robinson, B. H., Bañuelos, G. S., Conesa, H. M., Evangelou, M. W. H. and Schulin, R. (2009). The phytomanagement of trace elements in soil. Critical Reviews in Plant Sciences, 28(4), 240-266. Varrica, D., Dongarra, G., Sabatino, G. and Monna, F. (2003). Inorganic Geochemistry of Roadway Dust from the Metropolitan Area of Palermo, Italy. Environmental Geology, 44, 222-230. Viklander, M. (1998). Particle size distribution and metal content in street sediments. Journal of Environmental Engineering, 124, 761-766.  192  APPENDICES  APPENDIX A - PRELIMINARY STUDIES  Main Site  Background site  Figure A.1 Satellite Photograph (HW1 - Study site)  Main Site  Background site  Figure A.2 Satellite Photograph (HW17 - Study site)  193  Map of Canada  Figure A.3 Location of study site (HW 1)  Location of study site (HW 17)  194  Figure A.4 HW 1 study site  Figure A.5 HW 17 study site  195  Figure A.6 Plants collected from the HW1 study site in winter  Figure A.7 Plants collected from the HW1 study site in summer 196  Lamium purpureum  Plantago lanceolata  Agrostis exerata  Ranunculus repens  Ranunculus occidentalis Figure A.8  Holcus lanatus  Plants collected from the HW17 study site in summer 197  Table A.1. Summary of Trans-Canada Highway characteristics in Surrey, B.C and Highway 17 characteristics in Delta, B.C. Characteristics Type  Average Daily Traffic (ADT) Drainage area Surface pavement Number of lanes/direction Type of section Surrounding land use  Trans-Canada Hwy (HW 1) Mixed residential, industrial and parkland 82,900 vehicles (WestboundWB) 73,100 vehicles (EastboundEB) 500 m2 Asphalt 3 Elevated flush shoulder Agricultural, residential  Highway 17 Rural 20,417 vehicles (NorthboundNB) 22,899 vehicles (SouthboundSB) 1286m2 (NB) 1368m2 (SB) Asphalt 2 Elevated flush shoulder Agricultural  198  APPENDIX B - STAGE 1 STUDY  Stage 1 - Experimental Design 1  2  3  4  21  22  23  24  41  42  43  44  P1T1R1S1  P1T1R2S1  P1T1R1S2  P1T1R2S2  P1T2R1S1  P1T2R2S1  P1T2R1S2  P1T2R2S2  P1T3R1S1  P1T3R2S1  P1T3R1S2  P1T3R2S2  5  6  7  8  25  26  27  28  45  46  47  48  P2T1R1S1  P2T1R2S1  P2T1R1S2  P2T1R2S2  P2T2R1S1  P2T2R2S1  P2T2R1S2  P2T2R2S2  P2T3R1S1  P2T3R2S1  P2T3R1S2  P2T3R2S2  9  10  11  12  29  30  31  32  49  50  51  52  P3T1R2S1  P3T1R2S1  P3T1R1S2  P3T1R2S2  P3T2R2S1  P3T2R2S1  P3T2R1S2  P3T2R2S2  P3T3R2S1  P3T3R2S1  P3T3R1S2  P3T3R2S2  13  14  15  16  33  34  35  36  53  54  55  56  P4T1R1S2  P4T1R2S1  P4T1R1S2  P4T1R2S2  P4T2R1S2  P4T2R2S1  P4T2R1S2  P4T2R2S2  P4T3R1S2  P4T3R2S1  P4T3R1S2  P4T3R2S2  17  18  19  20  37  38  39  40  57  58  59  60  P5T1R2S2  P5T1R2S1  P5T1R1S2  P5T1R2S2  P5T2R2S2  P5T2R2S1  P5T2R1S2  P5T2R2S2  P5T3R2S2  P5T3R2S1  P5T3R1S2  P5T3R2S2  Plants P1 – Brassica napus P2 – Helianthus annuus P3 – Lolium perenne P4 – Festuca rubra P5 – Poa pratensis  61  62  71  72  81  82  P1T1R3S1  P1T1R3S2  P1T2R3S1  P1T2R3S2  P1T3R3S1  P1T3R3S2  63  64  73  74  83  84  P2T1R3S1  P2T1R3S2  P2T2R3S1  P2T2R3S2  P2T3R3S1  P2T2R3S2  65  66  75  76  85  86  P3T1R3S1  P3T1R3S2  P3T2R3S1  P3T2R3S2  P3T3R3S1  P3T3R3S2  67  68  77  78  87  88  P4T1R3S1  P4T1R3S2  P4T2R3S1  P4T2R3S2  P4T3R3S1  P4T3R3S2  69  70  79  80  89  90  P5T1R3S1  P5T1R3S2  P5T2R3S1  P5T2R3S2  P5T3R3S1  P5T3R3S2  Treatments T1 – Initial Soil T2 – Soil spiked with A level metals T3 – Soil spiked with C level metals  Replications : R1, R2, R3  Stage 1 - Experimental Design Plants 1. 5. 9. 13. 17.  P1T1R1S1 P2T1R1S1 P3T1R2S1 P4T1R1S2 P5T1R2S2  2. 6. 10. 14. 18.  P1T1R2S1 P2T1R2S1 P3T1R2S1 P4T1R2S1 P5T1R2S1  3. 7. 11. 15. 19.  P1T1R1S2 P2T1R1S2 P3T1R1S2 P4T1R1S2 P5T1R1S2  4. 8. 12. 16. 20.  P1T1R2S2 P2T1R2S2 P3T1R2S2 P4T1R2S2 P5T1R2S2  21. 25. 29. 33. 37.  P1T2R1S1 P2T2R1S1 P3T2R2S1 P4T2R1S2 P5T2R2S2  22. 26. 30. 34. 38.  P1T2R2S1 P2T2R2S1 P3T2R2S1 P4T2R2S1 P5T2R2S1  23. 27. 31. 35. 39.  P1T2R1S2 P2T2R1S2 P3T2R1S2 P4T2R1S2 P5T2R1S2  24. 28. 32. 36. 40.  P1T2R2S2 P2T2R2S2 P3T2R2S2 P4T2R2S2 P5T2R2S2  41. 45. 49. 53. 57.  P1T3R1S1 P2T3R1S1 P3T3R2S1 P4T3R1S2 P5T3R2S2  42. 46. 50. 54. 58.  P1T3R2S1 P2T3R2S1 P3T3R2S1 P4T3R2S1 P5T3R2S1  43. 47. 51. 55. 59.  P1T3R1S2 P2T3R1S2 P3T3R1S2 P4T3R1S2 P5T3R1S2  44. 48. 52. 56. 60.  P1T3R2S2 P2T3R2S2 P3T3R2S2 P4T3R2S2 P5T3R2S2  61. 63. 65. 67. 69.  P1T1R3S1 P2T1R3S1 P3T1R3S1 P4T1R3S1 P5T1R3S1  62. 64. 66. 68. 70.  P1T1R3S2 P2T1R3S2 P3T1R3S2 P4T1R3S2 P5T1R3S2  71. 73. 75. 77. 79.  P1T2R3S1 P2T2R3S1 P3T2R3S1 P4T2R3S1 P5T2R3S1  72. 74. 76. 78. 80.  P1T2R3S2 P2T2R3S2 P3T2R3S2 P4T2R3S2 P5T2R3S2  P1 – Brassica napus P2 – Helianthus annuus P3 – Lolium perenne P4 – Festuca rubra P5 – Poa pratensis  Treatments T1 – Initial Soil T2 – Soil spiked with A level metals T3 – Soil spiked with C level metals Replications : R1, R2, R3 81. 83. 85. 87. 89.  P1T3R3S1 P2T3R3S1 P3T3R3S1 P4T3R3S1 P5T3R3S1  82. 84. 86. 88. 90.  P1T3R3S2 P2T2R3S2 P3T3R3S2 P4T3R3S2 P5T3R3S2  Figure B.1 5*3*3 = 45 pots. Two sets for destructive sampling at 90 and 120 DAS (45*2 = 90). T1 – B0, T2 – BA, T3 – BC. S1 – 90 DAS, S2 – 120 DAS. 199  Table B.1. Sequential chemical extractions for the partitioning of metals and their respective reagents Fraction  Metal Species  1  Exchangeable  2  Carbonate  3  Oxide  Extracting Reagent 1 M KNO3 adjusted to natural soil pH 1 M NaOAc, adjusted to pH 5 with HOAc 0.04 M NH2OH⋅HCl in 25% (v/v) HOAc  Treatment o  25 C 1 hour agitation o  25 C 1 hour agitation o  96 C, 6 hours with intermittent agitation o  1) 85 C, 2 h intermittent agitation; 2) addition of (H2O2), 30% H2O2 (pH 2 w/ HNO3) + 0.02 M 3 h intermittent agitation; 4 Organic HNO3 3) 3.2 M (NH4OAc) in 20% (v/v) HNO3 continuous agitation for 30 minutes. HNO3/HCl Complete digestion 5 Residual Reference - Tessier et al (1979) as modified by Preciado and Li (2006) The solubility of metal fractions is in the order: exchangeable > carbonate specifically adsorbed > Fe-Mn oxide > organic > residual. Water-soluble and exchangeable forms of metals in soils are considered readily mobile and available to plants, whereas metals incorporated into crystalline lattices of clays (residual forms) appear to be relatively inactive. Metals, precipitated as carbonates, occluded in Fe, Mn, and Al oxides, and complexed with organic matter, could be strongly bound in soils, depending on the actual composition, physical and chemical properties of soil.  200  APPENDIX C - STAGE II STUDY  S tag e 2 - E xp erim ental en tal D esig n P lants - 3  R eplications : R 1, R 2, R 3  P 1 – Lolium P 2 – Festuca P 3 – P oa Treatm ents - 6 T1 – Initial soil alone T2 - Initial soil + A le vel spiking of m etal con tam in ants (C u, P b, M n and Zn) T3 – T 2 + Lim e T4 – T 2 + P T5 – T 2 + C om post T6 – T 2 + Lim e + P + C om post  Figure C.1  1. P 1T 1R 1  2. P1T 1R 2  3.  P 1T 1R 3  4. P1T 2R 1  5.  P1T 2R 2  6. P 1T 2R 3  7. P 1T 3R 1  8. P1T 3R 2  9.  P 1T 3R 3  10. P1T 4R 1  11.  P1T 4R 2  12. P 1T 4R 3  13. P 1T 5R 1  14. P1T 5R 2  15.  P 1T 5R 3  16. P1T 6R 1  17.  P1T 6R 2  18. P 1T 6R 3  19. P 2T 1R 1  20. P2T 1R 2  21.  P 2T 1R 3  22. P2T 2R 1  23.  P2T 2R 2  24. P 2T 2R 3  25. P 2T 3R 1  26. P2T 3R 2  27.  P 2T 3R 3  28. P2T 4R 1  29.  P2T 4R 2  30. P 2T 4R 3  31. P 2T 5R 1  32. P2T 5R 2  33.  P 2T 5R 3  34. P2T 6R 1  35.  P2T 6R 2  36. P 2T 6R 3  37. P 3T 1R 1  38. P3T 1R 2  39.  P 3T 1R 3  40. P3T 2R 1  41.  P3T 2R 2  42. P 3T 2R 3  43. P 3T 3R 1  44. P3T 3R 2  45.  P 3T 3R 3  46. P3T 4R 1  47.  P3T 4R 2  48. P 3T 4R 3  49. P 3T 5R 1  50. P3T 5R 2  51.  P 3T 5R 3  52. P3T 6R 1  53.  P3T 6R 2  54. P 3T 6R 3  Layout of the stage II experiment (6*3*3 = 54 pots. Stage of sampling – 90 DAS)  201  Application of amendments  Lime - The recommended dose of lime was 10 tons/ha from the lime requirement estimated (McLean, 1982). Dolomite (finely ground) was used as the liming material. CaCO3 (52.48 %) MgCO3 (40.96 %) Neutralizing value as CaCO3 equivalent (101 %) Screen size – 100 % passing 10 MESH (1.70 mm) 80 % passing 20 MESH (0.85 mm) 30 % passing 100 MESH (0.15 mm). Phosphate – The recommended dose was 135 kg P2O5/ha, based on the available P status of the soil (10.5 ppm). The source of P used was Ca HPO4. 2H2O (41 % P2O5). Compost - City of Vancouver Yard Trimming Compost (pH - 6.4; electrical conductivity - 3.2 dSm-1; C/N ratio - 21.3, Cu – 1.2 mg/kg, Zn - 42 mg/kg, Fe – 61 mg/kg and Mn - 146 mg/kg) was used. The recommended dose was 10 tons/ha based on the organic matter status of the soil. McLean, E. O. (1982). Soil pH and Lime requirement. In: Page, A.L., Miller, R.H. and Keeney, D. R. eds. Methods of Soil Analysis. Madison, Wisconsin USA. p. 214.  202  Figure C.2  One month after sowing  Figure C.3  Two months after sowing  Harvest stage (Three months after sowing)  203  Figure C.4  204  Table C.1. Summary of weather data at UBC from May 2006 to December, 2006, during pot experiments. Month May, 2006 June, 2006 July, 2006 August, 2006 September, 2006 October, 2006 November, 2006 December, 2006  Mean temperature (°C) 12.6 15.6 17.9 17.1 14.9 10.1 5.5 4.6  Table C.2. Root and Shoot biomass, dry weight (DW) (g/pot) Treatments Root weight (g/pot) Lolium B0 0.54bc BA 0.52c BAL 0.68ab BAP 0.74a BAO 0.50c BALPO 0.77a  Average humidity (%) 81 84 83 87 89 93 96 96  Shoot weight (g/pot) 0.79c 0.71c 1.00b 1.26ab 1.4a 1.50a  Total biomass (g/pot) 1.33 1.23 1.68 2 1.9 2.27  Festuca  B0 BA BAL BAP BAO BALPO  0.51c 0.28d 0.49c 0.58b 0.57bc 0.61bc  0.63cd 0.42e 0.87c 1.02b 1.15b 1.25ab  1.14 0.7 1.36 1.6 1.72 1.86  Poa  B0 BA BAL BAP BAO BALPO F  0.63b 0.50 0.63b 0.64b 0.74a 0.76a *  0.59d 0.52d 0.68cd 1.17b 1.28ab 1.32a *  1.22 1.02 1.31 1.81 2.02 2.08 *  Mean values, n = 3. * F significant at P<0.05. Significantly different statistical values (P<0.05) according to the Least Significance Test in each column are followed by different letters. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.  205  APPENDIX D - STAGE III STUDY  Figure D.1 Experimental Design Stage III  206  50 cm v  Ist sampling  1m  50 cm  2nd sampling 3rd sampling  1m Figure D.2 Lay out of each plot Application of amendments  Lime - The recommended dose of lime was 10 tons/ha from the lime requirement estimated. Dolomite (finely ground) was used as the liming material. Phosphate – The recommended dose was 135 kg P2O5/ha, based on the available P status of the soil. The source of P used was Ca HPO4 2H2O (41 % P2O5). Table D.1. Summary of weather data at Tsawwassen, Deltaport Way from May 2007 to February, 2008, during the field experiment.  Month May, 2007 June, 2007 July, 2007 August, 2007 September, 2007 October, 2007 November, 2007 December, 2007 January, 2008 February, 2008  Mean temperature (°C) 12.8 15.2 18.8 17.8 14.2 9.6 5.9 3.2 5.5 8.6  Total precipitation (mm) 37 80 53 8.4 73.6 155.2 116.2 181.6 137.6 68.6  207  Figure D.3 First sampling (90 days after sowing)  208  Figure D.4 Second sampling (180 days after sowing)  209  Figure D.5 Third sampling (270 days after sowing)  210  APPENDIX E - QA/QC. ABSTRACT OF ANOVA – FIELD EXPERIMENT  E.1. QA/QC  Pot experiments –Stage I and stage II experiments are conducted in CRD (Completely Randomized Design) with three replications. After a thorough mixing of treatments with soil, the moisture content was maintained at field capacity by taking weight of pots and adding the required amount of water. Randomization of pots to ensure uniform distribution of growth conditions was done every week. For stage 1 experiment, there were two stages of sampling, 90 and 120 DAS to find out the metal partitioning and metal removal at two different stages of plant growth. Since Brassica flowered first and the maximum flowering was at 90 DAS and senescence at 120 DAS, all samples were collected at the same time for consistency and comparability. For stage II experiment, there was only one sampling stage since the aim was to evaluate the effect of soil amelioration in influencing the plants to stabilize soil metals. Field experiment - The design for the field experiment was Completely Randomized Factorial Experiment in Split Plot Design with three replications. Soil and plant samples were collected from all plots at 90, 180 and 270 DAS, corresponding summer, autumn and winter. Each plot was demarcated to three portions with flag stakes for uniform sampling during three different seasons. E.1.1 Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer  QA/QC procedures and protocols consisted of 1 replicate analysis in 20 samples, method blanks, blank spikes and matrix spikes, 1 per batch. Multipoint calibrations were performed daily and verified every 20 samples, with a tolerance of ± 10% of initial calibration. The accuracy of the methodology was evaluated by analyzing two certified reference soil samples (CSSC-1 and CSSC- 2).  211  E.1.2. Standard reference soil samples  Table E.1. Total metal concentrations (mg/kg) (n = 3) Obtained values Soil Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) CSSC1 48.6 28.4 1398.9 CSSC2 49.2 17.2 845.4 Reported values Soil Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) CSSC1 42 24 1403 CSSC2 43 12 874 (Mckeague, J. A.,Shedrick, B. H. and Desjardins, J. G. (1978). Compilation reference soil samples. Soil Research Institute, Ottawa).  Zn (mg/kg) 99.6 69.7 Zn (mg/kg) 91 75 of data for CSSC  212  E.1.3. Comparison of ∑SSE with total metal concentrations  Table E.2. Initial values, before plant growth (HW 1) Treatment  Exch. (mg/kg)  Oxide (mg/kg)  Organic (mg/kg)  B0 BA  2.1 3.7  11.6 22.9  17.6 28.1  B0 BA  3.1 9.3  40.9 50.9  12.7 19.4  Residual (mg/kg)  Total SSE Total (mg/kg) metal (mg/kg)  R.D (%)  31 34  63 89  52 80  6 11  28.1 34.7  84.8 115.3  93 146  11 4  78.1 80  207 370  223 418  13 8  20.2 54.5  82.4 170.4  70 148  7 8  Cu Pb Mn B0 BA  10.3 27.8  119.7 238.9  9.3 23.4 Zn  B0 BA n=3  9.2 34.9  38.7 62.8  14.3 18.2  213  Table E.3. Stage 1 (90 DAS) Treatments  Exch. (mg/kg)  Oxide (mg/kg)  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  2.0 1.3 0.6 1.0 0.5 0.7 1.4 1.3  11.7 23.8 12.3 19.6 10.9 10.6 24.3 10.1  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  3.3 6.37 2.5 4.0 2.4 1.8 3.9 1.7  27.4 50.5 29.7 45.8 33.1 27.2 42.9 29.9  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0  5.1 19.7 9.8 24.7 11.4 7.6 24.2 11.4  89.4 151.2 91.7 152.1 97.8 105.1 158.7 105.3  9 21 7 20 8 6 18 8  23 46 25 48 19 25 47 23  LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 n =3  Organic Residual (mg/kg) (mg/kg) Cu 18.6 15.6 26.9 25.8 15.1 22.2 25.3 26.2 15.7 21.1 18.9 15.1 21.5 23.5 13.4 18.1 Pb 29.3 38.3 47.5 40 22.1 35 33.4 48.1 25 23.3 25.6 27.7 44.6 38.7 24.6 26.7 Mn 29.6 65.0 77.4 119.4 29.1 56.6 57 117.8 33.3 52.9 28.0 48.7 50.5 117.9 29.6 49.8 Zn 18 26 44 34 18 30 39 46 14 15 15 27 37 27 16 28  Sum SSE  Total concn. (mg/kg)  R.D (%)  47.9 77.9 50.1 74.9 48.2 45.3 70.6 42.9  45 74 49 78 50 46 75 47  6 5 3 4 4 2 7 10  98.3 144.3 89.2 135.9 85.8 80.51 134.2 83  86 132 90 140 88 89 137 90  13 9 8 3 3 1 2 9  189 367 186 350 196 188 351 197  207 381 209 396 210 203 380 211  10 4 13 14 7 8 9 7  76 120 80 156 56 73 132 75  64 138 67 142 76 65 144 63  16 15 18 9 36 11 10 16  214  Table E.4. Stage II (soils grown with Poa pratensis) Treatments Exch.  R.D(%)  Total SSE  Total concn. (mg/kg)  26.3 15.3 18.3 23.7  79.2 64.5 69.7 76.9  85 74 78 79  7 13 11 3  51 72 44 71  126.5 122.5 114.79 122.25  140 134 133 142  10 9 14 15  78 52 59 51  377 310.9 309.2 357.3  386 331 307 390  2 6 0.1 9  65.7 55.8 30 47  160 123 119 156  142 106 137 139  13 16 14 12  SSE extraction (mg/kg) Oxide Organic Residual  BAL BAP BAO BALPO  1.8 5.7 2.0 0.9  25.1 20.6 19.9 16.5  BAL BAP BAO BALPO  1.10 0.60 0.19 0.05  56.9 43.8 30.1 27.3  BAL BAP BAO BALPO  7.6 19.7 14.7 6.4  282 231 216 284  BAL BAP BAO BALPO n=3  0.72 16 27.1 2.9  74.8 38.5 48.8 74.8  Cu 26.0 22.9 29.4 35.8 Pb 17.5 6.1 40.5 23.9 Mn 9.4 8.2 19.5 15.9 Zn 18.5 13.1 23.1 36.1  215  Table E.5. Stage III (T0 at 90 DAS) Exch. CO3 Oxide Organic Residual Total concn. R.D (%) Plants (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) ∑SSE (mg/kg) Cu P1 4.7 8.3 5.2 14.5 13.7 46.4 72.7 P2 6.1 5.9 11.6 13.7 15 52.3 67.8 P3 4 6.8 18.4 17.8 14.4 61.4 81.2 P4 6.2 4.7 12.9 17 23.9 64.7 91.9 Pb P1 2.9 5.6 18.3 35.2 15.1 77 87.3 P2 5.7 8 14.1 19.8 29.3 76.9 81.7 P3 0.6 5.5 18.7 44.9 17.6 87.3 93.8 P4 3.2 7.8 10.8 22.3 22.9 67 85.8 Mn P1 15.4 3.5 45.6 7.9 55.1 127.5 144 P2 12.3 5.1 54.7 11 67.7 150.8 126 P3 16.3 1.5 59.3 8.9 61.3 147.3 164 P4 14.3 3 47 12.8 60.9 138 132 Zn P1 23.5 6.9 28.9 48.9 48.5 156.7 182 P2 18.9 11.8 38.8 85.7 34.4 189.6 154 P3 19.7 18.7 28.6 80.1 42.7 189.8 161 P4 14.8 10.9 41.8 67.8 49.1 184.4 174 n = 3. P1 – Lolium, P2 – Festuca, P3 – Poa, P4 – Lolium + Festuca + Poa  36 23 24 29 12 6 7 22 11 20 10 5 14 23 18 6  216  E.2. Abstract of ANOVA – Field Experiment. E.2.1. Metal fractionation, total soil metal concentrations and plant metal (root and shoot) concentrations Table E.6. Cu df Source Treatments (T) Plants (P) Time (Ti) P*T T*Ti P*Ti P*T*Ti S.E of mean  1 3 2 3 2 6 6  Exch. Cu *  CO3 Cu *  Oxide Cu *  Organic Residual Total Cu Cu Cu * * NS  Root Cu *  Shoot Cu NS  * * * * * * 1.002  * * * NS * NS 1.66  * * * * * * 2.4  * * NS * * * 2.08  * * * * * * 13.3  * * * * * * 4.43  Root Pb *  Shoot Pb *  * * * * * * 6.56  * * * * NS * 3.20  * * * * * * 1.24  * * * NS * * 4.3  Table E.7. Pb df Exch. CO3 Oxide Organic Residual Total Source Pb Pb Pb Pb Pb Pb Treatments 1 * * * NS * * (T) Plants (P) 3 * * * * * NS Time (Ti) 2 * * * * NS * P*T 3 * * * * * * T*Ti 2 * * * * * * P*Ti 6 * * * * NS * P*T*Ti 6 * * * * NS * S.E of 0.74 1.79 3.70 4.37 6.79 5.48 mean • n = 3, *F values significant at P<0.05, NS – F values not significant.  217  Table E.8. Mn df Source Treatments (T) Plants (P) Time (Ti) P*T T*Ti P*Ti P*T*Ti S.E of mean  1 3 2 3 2 6 6  Exch. Mn NS  CO3 Mn *  Oxide Mn *  Organic Residual Total Mn Mn Mn * * NS  Root Mn NS  Shoot Mn *  NS * * NS NS NS 1.88  NS * * NS NS * 0.81  * * NS * * NS 4.11  * * * * * * 2.27  * * NS NS * * 18.18  * * * * * * 73.55  Exch. Zn NS  CO3 Zn *  Oxide Zn *  Organic Residual Total Zn Zn Zn NS * NS  Root Zn NS  Shoot Zn *  NS * * NS NS * 5.77  * * * * * * 3.63  * * * * * * 6.59  * * * NS * * 8.18  * * * * * * 8.98  * * * NS * * 11.6  * * * * * * 3.27  * * * * * * 8.23  Table E.9. Zn df Source Treatments (T) Plants (P) Time (Ti) P*T T*Ti P*Ti P*T*Ti S.E of mean  1 3 2 3 2 6 6  * NS * * * * 4.67  * * * * * * 16.34  E.2.2. Bulk Soil Vs Rhizosphere soil, Design – Completely Randomised Factorial Experiment. Metal fractionation and total soil metal concentrations. Table E 10. Cu df Exch. Cu CO3 Cu Oxide Cu Organic Source Cu Treatments (T) 1 * NS NS * Plants (P) 3 * * * * Soil (S) 1 NS * * * P*T 3 * * * * T*S 1 NS * * * P*S 3 * * * * P*T*S 3 * * * * S.E of mean 1.37 0.42 1.85 2.19 • n = 3, *F values significant at P<0.05, NS – F values not significant.  Residual Cu NS * * * NS * * 2.17  Total Cu NS * * * NS * * 4.67  218  Table E.11. Pb Source Treatments (T) Plants (P) Soil (S) P*T T*S P*S P*T*S S.E of mean  df  Exch. Pb  1 3 1 3 1 3 3  * * * * * * * 0.23  CO3 Pb NS * * * NS NS NS 0.69  Oxide Pb  df  Exch. Mn * * NS * NS * NS 1.42  CO3 Mn Oxide Mn NS * * * * * * * * * * * * * 0.48 2.26  NS * * * NS * * 1.79  Organic Pb NS * * * * * * 3.58  Residual Pb Total Pb NS * * * * * * 3.96  * * * * * * * 4.13  Organic Mn NS * * NS NS NS NS 2.96  Residual Mn * * * * NS * NS 2.09  Total Mn NS * * NS NS * NS 14.04  Table E.12. Mn Source Treatments (T) Plants (P) Soil (S) P*T T*S P*S P*T*S S.E of mean  1 3 1 3 1 3 3  Table E.13. Zn df Source Treatments (T) Plants (P) Soil (S) P*T T*S P*S P*T*S S.E of mean •  1 3 1 3 1 3 3  Exch. Zn CO3 Zn NS NS NS * * * * * NS NS NS * NS * 6.67 2.39  Oxide Zn Organic Zn * * * * NS * * * * * * * * * 5.30 8.18  Residual Zn Total Zn NS * * * * * NS 4.22  * * * * NS * * 11.8  n = 3, *F values significant at P<0.05, NS – F values not significant.  219  APPENDIX F  List of publications from thesis  Refereed journals 1. Padmavathiamma, P. K. and Li, L. Y. (2007). Phytoremediation Technology: Hyperaccumulation Metals in Plants. Water, Air, and Soil Pollution 184: 105–126. 2. Padmavathiamma, P. K. and Li, L. Y. (2008). Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation 11(6): 575-590. 3. Padmavathiamma, P. K. and Li, L. Y. (2009). Phytoremediation and its Effect on Mobility of Metals in Soil: a Fractionation Study. Land Contamination and Reclamation 17(2), 223-236. 4. Padmavathiamma, P. K. and Li, L. Y. (2009). Phytostabilisation – a sustainable remediation for zinc toxicity in soils. Water, Air, and Soil Pollution 9(3-4), 253-260. 5. Padmavathiamma, P. K. and Li, L. Y. (2009). Effect of amendments on phytoavailability and fractionation of copper and zinc in contaminated soil. International Journal of Phytoremediation. (Accepted: August, 2009). 6. Padmavathiamma, P. K. and Li, L. Y. (2010). Phytoavailability and fractionation of lead and manganese in contaminated soil following application of three amendments. Bioresource Technolgy doi:10.1016/j.biortech.2010.01.149. . Other refereed relevant contributions (e.g., papers in refereed conference proceedings.) 1. Padmavathiamma, P. K. and Li, L. Y. (2006) Metal Hyper-Accumulation in Plants - an Overview of Phytoremediation Technology. Proceedings. 59th Geo-technical Conference. October 1-4. 2. Padmavathiamma, P. K., Li, L. Y. and Lavkulich, L. (2007). Heavy metal contamination and potential of local plants for phytoremediation along Highways. 9th International Conference on Biogeochemistry of Trace Elements (ICOBTE), Beijing, China. 3. Padmavathiamma, P. K. and Li, L. Y. (2007). Comparative phytoremediation potential of five different plant species for Cu in soils of British Columbia. 60th Geo-technical Conference, Ottawa, Canada. October 21-24, 2007. 4. Padmavathiamma, P. K. and Li, L. Y. (2008). Sustainable remediation of Pb along Highways. International Conference on Waste Engineering and Management, CSCE-HKIE, Hong Kong, May 28-30, 2008.  220  5. Padmavathiamma, P. K. and Li, L. Y. (2008). Phytostabilization of metals in Highway soils. GREEN5 International Conference, Vilnius, Lithuania, July 1-4, 2008. 6. Padmavathiamma, P. K. and Li, L. Y. (2008). Cu fractionation and plant accumulation in a soil contaminated with Cu and amended with lime, compost, and organic matter. 61st Canadian Geotechnical Conference, Edmonton. September 21- 24, 2008. 7. Padmavathiamma, P. K. and Li, L. Y Y. (2008). Phytostabilisation– A sustainable remediation for Zn toxicity in soils. International Conference on Environmental Science and Technology (ICEST), Houston, Texas. July 28-31, 2008.  221  

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